Eur J Biochem 271, 3319–3329 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04265.x Relationships between structure, function and stability for pyridoxal 5¢-phosphate-dependent starch phosphorylase from Corynebacterium callunae as revealed by reversible cofactor dissociation studies Richard Griessler, Barbara Psik, Alexandra Schwarz and Bernd Nidetzky Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria Using 0.4 M imidazole citrate buffer (pH 7.5) containing 0.1 mM L-cysteine, homodimeric starch phosphorylase from Corynebacterium calluane (CcStP) was dissociated into native-like folded subunits concomitant with release of pyridoxal 5¢-phosphate and loss of activity The inactivation rate of CcStP under resolution conditions at 30 °C was, respectively, four- and threefold reduced in two mutants, Arg234fiAla and Arg242fiAla, previously shown to cause thermostabilization of CcStP [Griessler, R., Schwarz, A., Mucha, J & Nidetzky, B (2003) Eur J Biochem 270, 2126– 2136] The proportion of original enzyme activity restored upon the reconstitution of wild-type and mutant apo-phosphorylases with pyridoxal 5¢-phosphate was increased up to 4.5-fold by added phosphate The effect on recovery of activity displayed a saturatable dependence on the phosphate concentration and results from interactions with the oxyanion that are specific to the quarternary state Arg234fiAla and Arg242fiAla mutants showed, respect- ively, eight- and > 20-fold decreased apparent affinities for phosphate (Kapp), compared to the wild-type (Kapp  mM) When reconstituted next to each other in solution, apoprotomers of CcStP and Escherichia coli maltodextrin phosphorylase did not detectably associate to hybrid dimers, indicating that structural complementarity among the different subunits was lacking Pyridoxal-reconstituted CcStP was inactive but  60% and 5% of wild-type activity could be rescued at pH 7.5 by phosphate (3 mM) and phosphite (5 mM), respectively pH effects on catalytic rates were different for the native enzyme and pyridoxal-phosphorylase bound to phosphate and could reflect the differences in pKa values for the cofactor 5¢-phosphate and the exogenous oxyanion Structure–function relationship studies of a-glucan phosphorylases (GP) have a rich history in biochemical literature It is well established that pyridoxal 5¢-phosphate (PLP) is the essential cofactor in all known GPs [1] PLP is bound via a Schiff base between its aldehyde group and a conserved lysine side chain in the active site [1,2] The 5¢-phosphate group is a main catalytic component of PLP and is required for GP activity [2] The functional oligomeric state of GP is dimeric [3–5] It has been shown that dissociation of the subunits under localized denaturing conditions exposes PLP to solvent PLP is released from the enzyme and the activity is lost [6–8] Apo-phosphorylase can be reconstituted, either with PLP or a range of structural analogues thereof [2,9,10] Whereas restoration of enzyme activity upon the apofiholo conversion is determined by cofactor structure, the process of dimerization is relatively indiscriminate in respect to structural modifications of PLP Induction of structural complementarity of the interacting subunits such that they are able to recognize each other and associate to dimers is correlated with enzyme–cofactor bond formation [5,9] In a thorough investigation, Helmreich and colleagues prepared a series of hybrid phosphorylases in which one subunit contained PLP while the other was bound to an inactive cofactor analogue [5] They concluded that intersubunit contacts were also needed to elicit activity in a potentially active holo-monomer With very few exceptions [11,12], the results just summarized were obtained with a single enzyme, GP from rabbit muscle (RmGP) The activity of RmGP is under the control of allosteric and covalent regulatory mechanisms which are different or completely lacking in a large group of GPs from plants and microorganisms We therefore asked the question, what novel information might be gained by applying the same type of reconstitution experiments described for RmGP to another phosphorylase from a different source with different regulatory properties? While active-site residues are almost invariant in members of the GP family, the dimer interfaces have been quite variable during the evolution in respect to the specific interprotomeric contacts, as revealed by comparative 3D structural Correspondence to B Nidetzky, Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/ I, A-8010 Graz, Austria Fax: +43 316 873 8434, Tel.: +43 316 873 8400, E-mail: bernd.nidetzky@tugraz.at Abbreviations: GP, glycogen phosphorylase; EcMalP, Escherichia coli maltodextrin phosphorylase; CcStP, Corynebacterium callunae starch phosphorylase; PLP, pyridoxal 5¢-phosphate; PL, pyridoxal; RmGP, rabbit muscle GP Enzyme: a-glucan phosphorylase or a-1,4-D-glucan:orthophosphatea-D-glucosyltransferase (EC 2.4.1.1) (Received 25 March 2004, revised 21 June 2004, accepted 22 June 2004) Keywords: apo-phosphorylase; a-glucan; glycogen; maltodextrin; pyridoxal 5¢-phosphate Ĩ FEBS 2004 3320 R Griessler et al (Eur J Biochem 271) [13] and structure-based sequence analyses [14,15] The overall contact pattern at the subunit interfaces of different regulated and nonregulated GPs is however, well preserved [13] Thus one would like to know what directs subunit interactions towards the induction of full enzymatic activity and optimum stability in a dimer of phosphorylase This is a significant and central problem to the study of catalysis by GPs and oligomeric enzymes in general where the individual subunits seem to possess all of the requisite chemical functions but are in a catalytically inactive and unstable conformation The detailed examination of the steps involved in subunit dissociation and reassociation will contribute to a better understanding of the dimerization process per se and the role of interprotomeric contacts to generate a functional enzyme The utilization of a phosphorylase devoid of the complex regulatory mechanisms seen in RmGP allows the analysis to be strictly focused on catalytic activity and stability We chose starch phosphorylase from Corynebacterium callunae (CcStP), which has been characterized biochemically and structurally [15,16], for particular reason The intersubunit contacts stabilizing the functional CcStP dimer are strengthened by > 100-fold when oxyanions such as phosphate bind to this enzyme [17] Enzyme–oxyanion interactions occur at a protein site different from the active site, and thermostabilization is the result of a protein conformational change induced by the binding event Residues involved in the structural rearrangement are located within the predicted dimer contact region of CcStP [15] Reversible subunit dissociation experiments should thus be useful to explore structural requirements for the phosphate effect on CcStP stability We report here the preparation of apo-CcStP and the characterization thereof in respect to structural properties and kinetic stability The process of reconstitution with PLP has been analyzed using CcStP and four site-specific mutants in which amino acid replacements within the dimer contact region have led to altered oxyanion-dependent kinetic stabilities [15,18] The relative timing of steps involved in dimer formation and appearance of thermostabilization by phosphate has been examined The role of the cofactor 5¢-phosphate group in the induction of stability and stabilization of the CcStP dimer has been studied Subunit complementation experiments are reported which were designed to detect formation of possible hybrid dimers of CcStP and maltodextrin phosphorylase from Escherichia coli (EcMalP) Finally, we show results from kinetic studies of CcStP reconstituted with pyridoxal (PL), a cofactor analogue in which the original 5¢-O-PO32– group is replaced by 5¢-O-H Materials and methods Enzymes, substrates and other materials Recombinant CcStP and site-directed mutants thereof were produced as described elsewhere [15,18] Natural CcStP was purified by a reported procedure [16] If not stated otherwise, recombinant CcStP was used EcMalP was prepared according to Eis et al [19] Analytical enzymes and enzyme substrates were specified in previous papers [15–18] All other chemicals were of reagent grade and obtained from Sigma and Fluka Preparation of apo-Cc StP and apo-Ec MalP Screening for buffer conditions in which apo-CcStP could be prepared, led to selection of 0.4 M imidazole citrate and 0.1 M cysteine hydrochloride, in short, the resolution buffer Various pH values between 5.0 and 8.0 were tested, and a pH of 7.0 was chosen (see below) Prior to the resolution, CcStP and site-directed mutants thereof were doubly gel filtered using NAP or NAP 10 columns (Amersham Biosciences) to remove phosphate from storage stock solutions to an end concentration below 0.1 mM The enzymes were incubated in the resolution buffer at 30 °C using protein concentrations in the range 0.5–2.0 mgỈmL)1 until the residual activity was between 1.5 and 2.5% of the original level The resolution buffer was then replaced by a 50 mM triethanolamine buffer, pH 7.0, using gel filtration with a NAP column Separate control experiments for wild-type CcStP showed that the fourfold variation in protein concentration in our experiments was not an important factor of the rate of resolution Apo-EcMalP was prepared using a protocol developed by Palm and coworkers (D Palm, Theodor-Boveri-Institut fur Biowissenschaften, Universitat Wurzburg, Germany; ă ă ă personal communication) The enzyme was diluted to mgỈmL)1 in 50 mM Mes buffer, pH 7.0, containing 25 mM KCl and mM dithiothreitol An equal volume of M cysteine hydrochloride dissolved in the same buffer was added to give a final concentration of 0.5 M Resolution was obtained by adjusting the pH with HCl to a value of 5.05 at °C The enzyme was incubated under these conditions until the residual activity was about 1.5% of the original level Apo-EcMalP was precipitated by ammonium sulphate at 65% saturation, and the pellet was resuspended in 50 mM potassium phosphate buffer, pH 7.0 The time course of apo-phosphorylase formation was monitored by using a number of methods [17]: enzyme activity measurements using samples taken from the incubation mixture; column sizing experiments to determine the subunit association state of the protein; CD spectroscopic measurements; determination of protein-bound and dissociated PLP This latter measurement was performed after ultrafiltration of the sample using 30 kDa cut off microconcentrator tubes The PLP content of the protein-containing retentate was measured using both semiquantitative fluorometric analysis and a quantitative spectrophotometric test [17] The filtrate, which was devoid of protein, was the subject of quantitative analysis for PLP content Apo-phosphorylases were always prepared for immediate further use and not stored for longer than about h at °C Appropriate control measurements showed that the inactivation of apo-enzymes was not significant under these conditions Reconstitution of apo-phosphorylases Apo-phosphorylase of CcStP (about 0.1–0.4 mgỈmL)1) was brought to 50 mM triethanolamine buffer, pH 7.0, containing a concentration of potassium phosphate between < 0.05 and 80 mM PLP at a concentration of between 0.0 and 100 lM was added to reconstitute the holo-enzyme The reaction was carried out at 30 °C and typically, the time Ó FEBS 2004 Cofactor dissociation studies of starch phosphorylase (Eur J Biochem 271) 3321 course of recovery of enzyme activity was monitored up to 180 When addition of fresh PLP did not further enhance the regain of activity, reconstitution was considered to be exhaustive Reconstituted CcStP was characterized in respect to its structural properties using CD spectroscopy, cofactor fluorescence and analytical gel filtration using Superose 12 HR 10/30 (see below) Kinetic parameters of the direction of a-glucan phosphorolysis and synthesis were determined as described below Reconstitution of apoEcMalP was performed at 30 °C in 50 mM potassium phosphate buffer, pH 7.0, and incubation was carried on h after addition of 100 lM PLP Using the conditions described above, a reconstitution experiment was carried out in which apo-CcStP (0.35 mgỈmL)1 of the natural enzyme) and apo-EcMalP (1.35 mgỈmL)1) were incubated with 100 lM PLP next to each other in solution Therefore, heterodimerization would have been possible, and the aim was to either detect it or rule out its occurrence under the conditions used The protein solution was loaded on to a mL Econo-Pac column of ceramic hydroxylapatite type II (Bio-Rad) equilibrated with 50 mM potassium phosphate buffer, pH 6.8 Elution was carried out at room temperature with a step gradient of M potassium phosphate buffer, pH 6.8, at a flow rate of  40 cmỈh)1 Fractions containing protein were collected, concentrated using ultrafiltration microconcentrator tubes, and gel filtered using NAP 10 columns Characterization of the fractions was carried out in respect to: the N-terminal sequence determined by automated Edman degradation; stability at 50 °C when 0.3 M potassium phosphate (pH 7.0) was present; and kinetic parameters for phosphorolysis of maltohexaose (Sigma) at 30 °C Phosphorylase activity was measured in the direction of a-glucan phosphorolysis using a continuous, phosphoglucomutase and NAD+-dependent glucose 6-phosphate dehydrogenase-coupled spectrophotometric assay, described in more detail elsewhere [16] If not mentioned otherwise, maltodextrin 19.4 (Agrana, Gmund, Austria) was ă the a-glucan substrate Initial rates of a-glucan phosphorolysis and synthesis were recorded with discontinuous assays, as reported previously [16] Linear plots of product concentration vs time were converted into rates Kinetic parameters were obtained from nonlinear fits of initial rate data to Eqn (1) using the SIGMAPLOT program (SPSS Inc., Chicago, IL, USA), ð1Þ where v is the initial rate, kcat is the turnover number, [E] is the molar concentration of enzyme active sites (based on the stoichiometry of PLP and enzyme subunit), Km is an apparent Michaelis constant, and [S] is the substrate concentration When inhibition at high [S] was observed, Eqn (2) was used: v ẳ kcat ẵEẵS=Km ỵ ẵS ỵ ẵS2 =KiS ị log rate ẳ logẵC=1 ỵ Ka =ẵHỵ ị 3ị where C is the pH-independent value of the rate, Ka is a macroscopic acid dissociation constant, and [H+] is the proton concentration Equation (3) implies a pH profile that is level below pKa and decreases above pKa with a slope of )1 Stability of apo-phosphorylase Apo-phosphorylase ( 0.2 mgỈmL)1) was incubated in 0.1 M sodium acetate buffer, pH 6.9, at 22 °C At certain times between 0.2 and 20 h, samples were taken from the reaction mixture, PLP (40 lM) and potassium phosphate (50 mM) were added, and reconstitution was allowed to proceed for up to h before recovered enzyme activity was measured The activity of the reconstituted phosphorylase at zero incubation time served as the control A number of compounds were tested in respect to a potential stabilization of apo-phosphorylase, and they were added in the concentrations shown under Results Pyridoxin 5¢-phosphate was prepared by reduction of PLP with NaBH4 Control experiments were carried out in which pyridoxin 5¢-phosphate (2 mM) was incubated at 30 °C with apo-phosphorylase and regain of activity was recorded over time The total lack of recovery of activity proved that the reduction of PLP was complete Structural characterization Enzyme kinetic measurements v ẳ kcat ẵEẵS=Km ỵ ẵSị 8.0 If not indicated otherwise, it was proved that enzyme inactivation during the time of the discontinuous assay ( 15 min) was not a source of an observable pH dependence of activity pH profiles were fitted to Eqn (3), ð2Þ where KiS is the substrate inhibition constant pH effects of enzyme-catalyzed initial rates were recorded at 30 °C in 0.1 M sodium acetate buffer in the pH range 5.0– CD spectroscopic measurements were carried out with a Jasco J-600 spectropolarimeter using quartz cuvettes of 0.1 cm pathlength Spectra of protein samples ( 0.1 mgỈmL)1) were recorded at 23 ± °C in the range 200–240 nm If not mentioned otherwise, a 50 mM potassium phosphate buffer, pH 7.0, was used Column sizing experiments were carried out with Superose 12 HR 10/30 (22 mL bed volume) using a 50 mM potassium phosphate buffer, pH 7.0, containing 200 mM NaCl and 0.1% (w/v) NaN3 Approximately 200 lg of protein dissolved in 0.5– 1.0 mL of buffer were loaded on to the column, and elution of protein was detected at 280 nm using an ¨ Aktaexplorer system (Amersham Biosciences) Fluorescence measurements were performed with a Hitachi F-2000 spectrofluorometer using Hellma QS 101 cuvettes The excitation wavelength was set to 330 nm, and emission spectra were recorded in the range 360–600 nm Typically, a protein concentration of 0.4 mgỈmL)1 dissolved in triethanolamine buffer, pH 7.0, was used Results Preparation and characterization of apo-Cc StP Apo-CcStP was obtained at a practically useful rate by incubating CcStP in concentrations of between 0.5 and 2.0 mgỈmL)1 in 0.4 M imidazole citrate buffer, pH ¼ 6.8, Ĩ FEBS 2004 3322 R Griessler et al (Eur J Biochem 271) containing 0.1 M L-cysteine hydrochloride at 30 °C Loss of enzyme activity served as the reporter of formation of the apo-enzyme under these conditions Semi-logarithmic plots of the fraction of remaining active CcStP against time were linear, suggesting that inactivation can be approximated by a pseudo first-order model The half-life of the holophosphorylase was  60 at pH 7.0 The inactivation rate was pH-dependent and decreased at pH values below 6.5 No significant loss of activity was observed at pH 5.0– 5.5 over 1.5 h When 50 mM potassium phosphate or potassium sulphate was present in the buffer, pH 7.0, formation of apo-phosphorylase was not detected over a 24 h long incubation time, indicating a half-life of 100 h or greater Therefore, stabilization of the native dimer structure by the oxyanions must be > 100-fold (¼ 100/1), in good agreement with previous results on the thermostabilization of CcStP [15,17,18] Column sizing experiments revealed that the apo-phosphorylase is a monomer It does not contain bound PLP within limits of detection of the denaturing spectrophotometric assay (± 2%) It completely lacks the characteristic fluorescence emission of the cofactor in native CcStP which occurs in the wavelength range 480–560 nm (see later) Typically, apo-phosphorylases of CcStP and mutants thereof contained equal to 2% of the original enzyme activity which can be detected before and after the gel filtration to replace the resolution buffer Figure shows the time course of inactivation of apoCcStP at 22 °C in the absence and presence of potential stabilizers The half-life of apo-phosphorylase was approximately 15 h, and we observed only small effects on stability of added phosphate, sulphate, and the cofactor derivative pyridoxin 5¢-phosphate By contrast, UDP-a-D-glucose conferred substantial extra stability to CcStP ADP-a-Dglucose stabilized apo-CcStP to about the same extent as UDP-a-D-glucose (not shown) Gel filtration analysis of apo-CcStP was carried out under conditions in which UDPa-D-glucose (1 mM) was added to the elution buffer The apo-enzyme eluted as a single protein peak and with a retention time expected for a monomer of  90–100 kDa Therefore, the stabilizing effect of UDP-a-D-glucose is clearly not due to formation of an apo-oligomer induced by the binding of the nucleotide sugar The presence of maltopentaose (5 mM) resulted in a moderate 1.5-fold increase in the half-life of apo-CcStP Effects of mutations in the dimer contact region on the rate of apo-enzyme formation The pseudo first-order rate constants of inactivation in resolution buffer at pH 7.0 were determined for CcStP and five mutants thereof, using straight-line fits of the data plotted as logarithmic fraction of residual activity vs time The results are summarized in Table Comparison of rate constants shows that the effect of the mutation may be stabilizing (R234A, R242A), neutral (S238A, S224A), or destabilizing (R226A), compared to the wild-type Except for R226A and R242A mutants (Table 1), all enzymes were stable for h in the presence of mM potassium phosphate and potassium sulphate Reconstitutions with PLP of apo-Cc StP and mutants thereof, and characterization of the wild-type holo-enzyme Incubation of apo-CcStP (0.2 mgỈmL)1; 2.2 lM enzyme subunits) at 30 °C in 50 mM triethanolamine buffer, pH 7.0, containing 50 mM potassium phosphate led to a gradual regain of enzyme activity in a PLP concentration-dependent manner Nine levels of PLP between and 100 lM were tested, and the activity recovered after a 90 incubation (which was shown to be exhaustive) displayed a saturatable dependence on [PLP], with half-saturation being attained at KPLP ¼ 19 ± lM The recovery of activity when no PLP was added was not significant within the experimental error (± 1–2%) To prevent nonspecific reactions of the aldehyde group of PLP with protein lysines other than Lys634, a concentration of 2· KPLP was chosen for standard reconstitution Column sizing experiments revealed that reconstituted CcStP existed exclusively as a dimer CD and cofactor fluorescence emission spectra of native and reconstituted Table Half-lives (t1/2) of CcStP and mutants thereof in the resolution buffer at 30 °C and pH 7.0 Stable, no inactivation with h of incubation t1/2 (min) Fig Stability and stabilization of apo-CcStP The apo-enzyme ( 0.2 mgỈmL)1) was incubated at 22 °C in 0.1 M sodium acetate buffer, pH 6.9 Incubations were carried out without additive (d); mM potassium phosphate (s); mM sodium sulphate (.); mM pyridoxin 5¢-phosphate (,); and mM UDP-a-D-glucose (j) Activity in samples taken at the times indicated was measured after reconstitution with 40 lM PLP and 50 mM potassium phosphate as described under Materials and methods Protein No oxyanion mM Sulphate mM Phosphate Wild-type S224A R226A R234A S238A R242A 57 40 11 260 36 190 Stable Stable 100 ± 10.5 Stable Stable Stable Stable Stable 30 ± Stable Stable 300 ± 20 ± ± ± ± ± ± 4 0.5 25 10 Ó FEBS 2004 Cofactor dissociation studies of starch phosphorylase (Eur J Biochem 271) 3323 CcStP and apo-CcStP are shown in Fig The CD spectra of the three proteins are very similar overall, indicating similarity in respect to the relative composition of secondary structural elements However, the characteristic minima in ellipticity at 208 nm and 222 nm have greater intensities in the native enzyme, suggesting partial loss of a-helical structure in apo-CcStP and reconstituted holo-CcStP Data presented in Fig 2B proves that PLP is incorporated into apo-CcStP during reconstitution However, the intensity of cofactor fluorescence in the reconstituted enzyme is approximately 65% that observed in CcStP, and this difference agrees with differences in specific activities of Fig Comparison of spectral properties of native CcStP, apo-CcStP, and reconstituted enzyme using CD (A) and fluorescence (B) Spectra were recorded using approximately the same protein concentration (0.1 mgỈmL )1 ± 5%) in each case (A) Spectra of the native CcStP (j), the apo-CcStP (d), and the enzyme after exhaustive reconstitution in the presence of 100 lM PLP (,) (B) The fluorescence emission spectra are shown for native enzyme (––), apo-CcStP (ỈỈỈỈ), and reconstituted enzyme (- - -) The excitation wavelength was constant at 330 nm In (A) and (B), the reconstituted enzyme showed  65% of the original activity A 50 mM potassium phosphate buffer, pH 7.0, was used native and reconstituted phosphorylase Likewise, cofactor stoichiometry is decreased from a value of  in the wildtype to  0.6 in the reconstituted enzyme Apparent Michaelis constants of reconstituted CcStP were determined in 50 mM triethanolamine buffer, pH 7.0, for phosphate (4.0 ± 0.3 mM); and maltodextrin (3.9 ± 0.4 mM) in the direction of phosphorolysis; a-D-glucose 1-phosphate (1.0 ± 0.1 mM); and maltodextrin (33 ± mM) in the direction of synthesis After correction of turnover numbers for the fraction of active enzyme in holo-phosphorylase, native and reconstituted CcStP are not distinguishable in regard to their kinetic properties The time courses of recovery of enzyme activity upon reconstitution of wild-type and mutant apo-phosphorylases with 40 lM PLP were biphasic During the initial burst phase which was complete within min, there appeared up to 80% of the total enzyme activity recoverable under the conditions In the second phase, enzyme activity increased slowly to its final level and eventually decreased again Figure shows typical profiles of regain of activity vs time of reconstitution, obtained with the R226A mutant in the absence and presence of potassium phosphate In all cases except for the R242A mutant, the yield of enzyme activity (compared to the original level before resolution and expressed as a percentage thereof) was increased by added phosphate (Table 2) The effect of phosphate was composed of two components: first, a shift of apparent equilibrium for the reconstitution reaction towards the active enzyme and second, a stabilization of the reconstituted holo-enzyme against inactivation (which was shown to be irreversible) We compared recovery of activity of the wild-type under conditions in which phosphate (50 mM) was present from the beginning of the reconstitution or was added at the end of the burst phase (5 min) The yield was the same in both experiments within the experimental error The recovery of activity showed a saturatable dependence on the phosphate concentration Half-saturation constants for phosphate (KdPi) were obtained from nonlinear fits of values of final Fig Reconstitution of apo-enzyme of R226A mutant The assays contained 0.22 mgỈmL)1 protein and used 40 lM PLP Other conditions are reported under Materials and methods The symbols show the different concentrations of phosphate in mM, as indicated Ó FEBS 2004 3324 R Griessler et al (Eur J Biochem 271) Table Effect of phosphate on recovered enzyme activity during reconstitution of apo-enzymes of wild-type CcStP and mutants thereof with 40 lM PLP A 50 mM triethanolamine buffer, pH 7.0, was used KdPi is the half-saturation constant for phosphate The protein concentrations used varied in the range 1–4 lM of apo-enzyme (90 kDa) and were ‡ 10· the concentration of cofactor Control experiments carried out with the wild-type showed that the yield of reconstituted enzyme activity did not change as result of this variation in protein concentration The values in parentheses show the yield of recovered enzyme activity when no phosphate was present ND, not determined, because no significant dependence of recovered enzyme activity on [phosphate] was seen in the range 0–80 mM Protein KdPi (mM) Recovered enzyme activity (%) Wild-type S224A R226A R234A R242A 6.07 2.89 10.4 47.1 ND 66 59 85 68 56 ± ± ± ± 1.29 0.51 3.8 4.5 (37) (35) (48) (15) (48) recovered activity to Eqn (4) and are summarized in Table They reveal marked decreases in the apparent affinities of the R234A and R242A mutants for phosphate, compared to wild-type DEA ¼ DEAmax ẵPi =KdPi ỵ ẵPi ị 4ị where DEA is the difference in recovered enzyme activity in the presence and absence of phosphate, and DEAmax is the maximum value for DEA when phosphate is saturating Reconstitutions of apo-Cc StP and apo-EcMalP next to each other in solution Figure shows fractionation by hydroxylapatite chromatography of a protein mixture obtained by reconstitutions of apo-CcStP and apo-EcMalP under conditions that might enable subunit complementation to form a hybrid phosphorylase Through elution with an increasing phosphate concentration, two major fractions A and C were isolated which together accounted for more than 95% of the total protein loaded on to the column It is noteworthy that fractions A and C eluted exactly as expected for native CcStP and EcMalP, respectively Likewise, CcStP and EcMalP prepared by reconstitution of the corresponding apo-phosphorylases independent of one another displayed  70% of their original phosphorylase activities and eluted exactly as the native enzymes did (data not shown) Figure shows that a minor fraction B was also obtained Like fractions A and C, it contained phosphorylase activity Control experiments showed that under the conditions used, the fractionation of reconstituted EcMalP may yield a small fraction B depending on the applied amount of protein Protein fractions A–C were characterized functionally and structurally, as summarized in Table Production and characterization of PL-reconstituted CcStP PL could replace PLP in the reconstitution of apo-CcStP The formation of PL-phosphorylase after an exhaustive incubation time of  h showed a saturatable dependence on PL concentration, the optimum level of PL being approximately 250 lM Addition of PLP (40 lM) after a h incubation of apo-CcStP (0.3 mgỈmL)1) in the presence of PL (250 lM) did not restore further enzyme activity, suggesting that reconstitution with PL was complete PL-phosphorylase was as stable as the native enzyme or PLP-reconstituted CcStP at 60 °C in 300 mM potassium phosphate buffer, pH 7.0 Therefore, the cofactor phosphate group is not a component of oxyanion-dependent thermostabilization of CcStP When assayed in the direction of a-glucan synthesis at 30 °C (using conditions described in Fig 5), PL-phosphoryTable Characterization of protein species obtained through chromatographic fractionation of a mixture of apo-CcStP and apo-EcMalP reconstituted with 100 lM PLP next to each other in solution Figure gives details of the fractionation Fractions are labeled according to Fig KmG6 and KiG6 were obtained from nonlinear fits to Eqn (2) of the initial rate data recorded at a constant saturating concentration of 50 mM Pi KmG6 and KiG6 are the apparent Michaelis constant and the substrate inhibition constant for maltohexaose, respectively Half-life (t1/2) incubations were carried out at 50 °C in 300 mM potassium phosphate buffer, pH 7.0 Fraction A KmG6 (mM) KiG6 (mM) t1/2 (min) N-terminal sequence Properties of Fig Fractionation by hydroxylapatite chromatography of a protein mixture obtained by reconstitution of apo-CcStP and apo-EcMalP The protein elution profile, recorded by absorbance at 280 nm, is shown The dashed line indicates the elution gradient used See Materials and methods for details a Fraction B Fraction C 2.65 ± 0.35 360 ± 130 Stable P-E-K-Q-P-L-P-A-Aa 0.71 ± 0.07 31.8 ± 3.1 17 X-Qb 0.76 ± 0.10 21.9 ± 2.3 18 (S)-Q-P-(I)c CcStP EcMalP EcMalP Residue Ser1 is processed off in CcStP isolated from the natural organism [15,16] b X is an unidentified amino acid c Determination of the N-terminal sequence of fraction C was not completely clear at positions and Ó FEBS 2004 Cofactor dissociation studies of starch phosphorylase (Eur J Biochem 271) 3325 displayed saturatable concentration dependence, and half-maximum activation was observed at 0.5 mM At pH 7.5, about 57% of the wild-type level of activity could be recovered The Michaelis constant of the PL-enzyme for a-D-glucose 1-phosphate in the presence of phosphite was approximately 10 times that of CcStP The pH-dependence of activity under conditions of saturation in both substrates was determined for CcStP and PL-phosphorylase in the pH range 5.0–8.0 Initial rates were recorded in the directions of a-glucan phosphorolysis and synthesis, and assays for PL-phosphorylase in the synthesis direction contained a saturating level of activating phosphate (2.5 mM) Results are shown in Fig In either direction of reaction, enzymatic rates which are effectively turnover numbers (kcat) decreased at high and low pH Optimum catalytic rates for phosphorolysis were found at around pH 7.0 for both the native enzyme and PL-phosphorylase In the low pH region the pH profile of kcat for PL-phosphorylase was displaced outward by  1.0 pH unit, relative to the corresponding pH profile for CcStP The decrease in kcat (phosphorolysis; kpho) at high pH was similar for both enzymes In the synthesis direction, optimum conditions for kcat (ksyn) were observed at pH 6.0 for CcStP PLenzyme bound to phosphate showed maximum activity at pH 6.5–7.0 The pH profile of ksyn for PL-phosphorylase in the presence of phosphate was displaced outward by  1.0 pH units at high pH, compared to the pH profile of ksyn for wild-type CcStP Fits of the data to Eqn (3) yielded pKa values of 6.9 ± 0.3 and 7.9 ± 0.3 for wildtype enzyme and PL-CcStP, respectively Discussion Formation and characterization of apo-CcStP Fig Restoration of enzyme activity in PL-CcStP by exogenous (A) phosphate and (B) phosphite Incubations were carried out at 30 °C in 0.1 M sodium acetate buffer, pH 7.6, containing  30 lgỈmL)1 protein The substrate levels were constant at 80 gỈL)1 maltodextrin and 50 mM a-D-glucose 1-phosphate The levels of exogenous activator oxyanion are indicated by symbols and given in mM In (A) the concentrations of released phosphate were sufficient to allow an accurate determination of the activity in spite of the added phosphate The possible inhibition of the enzymatic reaction by phosphate is compensated using a high concentration of a-D-glucose 1-phosphate lase was inactive within the limits of detection of the experimental procedures Addition of phosphate or phosphite restored phosphorylase activity, as shown in Fig 5A,B, respectively The time course of formation of phosphate was linear when phosphate was used as the activator oxyanion The chosen level of phosphate (2.5 or mM) did not influence the enzymic rate significantly When phosphite was the activator oxyanion, the observed time courses were concave upward, perhaps indicating an autocatalytic effect of the released phosphate The reaction rate recorded at an oxyanion concentration of mM was  4.4 times higher with phosphate than phosphite Table summarizes the kinetic characterization of PL-CcStP The restoration of activity in PL-phosphorylase by phosphate A number of studies have identified prerequisites for reversible conversion of holo-GP into the apo-enzyme [2]: localized reversible denaturation promoting subunit dissociation; resolution of PLP through aldehyde-reactive compounds; and prevention of subunit aggregation In spite of these common characteristics, completely different protocols were needed for successful preparation of apo-enzymes of RmGP [2], Solanum tuberosum (potato tuber) starch phosphorylase [11], and EcMalP (D Palm, unpublished data) Apo-CcStP was obtained under conditions comparable to the ones used by Shaltiel et al [6] for resolution of RmGP; i.e using imidazole citrate and L-cysteine as structure-deforming and PLP-resolving reagents, respectively Interestingly, however, the pH dependence of the rate of resolution was opposite in the two enzymes, CcStP being stable under the slightly acidic conditions It was proposed by others [6–8] that the imidazolium ion is required for optimum resolution of RmGP at pH  6.0 In CcStP, imidazole obviously assists in locally disrupting the native structure but there was no evidence that its protonated form would be particularly effective Mutations within the dimer contact region of CcStP (Table 2; also [15,18]) had strong effects on the half-life of activity in resolution buffer Likewise, cofactor resolution was inhibited completely in the presence of phosphate or sulphate These results are in good agreement with the notion that weakening Ó FEBS 2004 3326 R Griessler et al (Eur J Biochem 271) Table Kinetic characterization of PL-CcStP in the presence of activator oxyanion Initial rates were recorded in 50 mM Tris-acetate buffer, pH 7.5, using a discontinuous assay in which samples were taken after 20, 40 and 60 of incubation The rates were calculated from linear plots of [Pi] released against the reaction time When phosphate was the activator oxyanion, initial rates were calculated from the difference between the concentrations of total phosphate at a certain incubation time and phosphate initially present In all cases this difference was sufficient to allow accurate determination of the enzymatic rate The values of vmax for the native phosphorylase determined in the presence and absence of 10 mM phosphite were identical within the experimental error, indicating weak (if any) inhibition by the added oxyanion Glc1P, a-D-glucose 1-phosphate; MD, maltodextrin (dextrin equivalent 19.4) Glc1P (mM) or MD (gỈL)1) PL-phosphorylase 1.0–50/80 50/5–120 50/80 Native phosphorylase 1.0–50/80 Activator oxyanion (mM) vmax (mg)1) Km (mM) Phosphite (10) Phosphite (10) Phosphate (0.1–3.0) 1.86 ± 0.11 1.95 ± 0.12 8.6 ± 0.2 11.2 ± 2.0 12.2 ± 2.7 0.49 ± 0.04 Phosphite (10) 15.0 ± 0.2 1.08 ± 0.11 subunit-to-subunit interactions in CcStP [15,17,18] is a key factor driving the resolution of the holo-enzyme Like apo-RmGP, apo-CcStP is monomeric and displays no enzyme activity A number of observations indicate that it retains native-like tertiary structure Stabilization of apoCcStP by UDP-a-D-glucose and ADP-a-D-glucose is particularly relevant because it suggests the preservation of a cofactor–substrate binding scaffold in apo-CcStP The nucleotide-activated sugars structurally resemble the noncovalent complex of PLP and a-glucose 1-phosphate that is formed at the phosphorylase active site in the course of the enzymatic reaction [20,21] The available evidence from gel filtration analysis excludes the occurrence of a transient apo-dimer lacking phosphorylase activity, induced by the presence of the stabilizing UDP-a-D-glucose UDP-aD-glucose at a level of mM inhibits the reaction of native CcStP to less than 15%, suggesting the absence of a highaffinity effector site for nucleotide sugars in the active holophosphorylase dimer Furthermore, it does not retard the resolution of the cofactor in CcStP (data not shown), indicating that the observed stabilizing effect is specific to the apo-enzyme Now, given that PLP resolution caused only minor denaturation of CcStP tertiary structure, it was especially interesting that thermostabilization of the holo-enzyme by phosphate was lost in apo-CcStP; and recovered fully upon reconstitution This result could indicate that in apo-CcStP (a) the actual oxyanion binding site was disrupted, or (b) a conformational change that accompanies oxyanion binding in the holo-enzyme cannot take place Whatever was truly responsible, the data suggest that dimerization is required for restoration of oxyanion-dependent thermostabilization of CcStP (see below) Reconstitution of the holo-enzyme Fig pH profiles in the direction of a-glucan synthesis (A) and phosphorolysis (B) catalyzed by wild-type CcStP (d) and PL-CcStP (s) activated by exogenous phosphate ions (A) Results were obtained in 0.1 M sodium acetate buffer containing 2.5 mM Pi The substrate levels were 80 gỈL)1 maltodextrin and 50 mM a-D-glucose 1-phosphate Solid lines are nonlinear fits of the data to Eqn (3) For PL-CcStP the catalytic rate at pH was not included in the calculation because its value reflects the effects of pH on both rate and enzyme stability (B) Results were obtained in 50 mM potassium phosphate buffer containing 80 gỈL)1 maltodextrin The lines indicate the trend of the data Reconstitution experiments were designed to address two specific questions of phosphorylase recognition First, apo-phosphorylases of CcStP and EcMalP associate in solution to form hybrid dimers? Secondly, is there a role of interactions between protein and oxyanion during the apofiholo conversion of CcStP? Complementation of phosphorylase apo-protomers in solution has obvious advantages over working with immobilized subunits, as described by others [5,7] However, it Ó FEBS 2004 Cofactor dissociation studies of starch phosphorylase (Eur J Biochem 271) 3327 requires methods which select for true hybrids Mixtures of reconstituted CcStP and EcMalP were separated by using hydroxylapatite chromatography [19] Conditions were used in which a hybrid would be clearly detectable if it displayed intermediate binding properties, compared to wild-type CcStP (weak binding) and EcMalP (strong binding) The observed elution pattern from the hydroxylapatite column was not consistent with the formation of hybrids in substantial amounts However, a small protein fraction was detected that eluted before and after the peaks clearly assigned to native or reconstituted EcMalP and CcStP, respectively This fraction contained enzyme activity and obviously, it could be a phosphorylase hybrid Furthermore, we had to consider the possibility that heterodimers escape detection because the different subunits interact with hydroxylapatite independently of one another Therefore, the three protein fractions obtained (A–C) were characterized by N-terminal sequencing and two parameters of enzyme function distinguishing sensitively between CcStP and EcMalP: (a) apparent substrate affinity and substrate inhibition in the direction of phosphorolysis of maltodextrins; and (b) kinetic stability at 50 °C The results showed that, within limits of detection of the fractionation procedure (5%), only wild-type enzymes were present and no hybrid dimers formed The observed small protein peak (fraction B) very likely contains reconstituted EcMalP, and its occurrence can be explained by an incomplete retention of reconstituted EcMalP by the hydroxylapatite column It seems that the structural complementarity between protomers of CcStP and EcMalP was not sufficient for the different subunits to recognize each other This finding is interesting because the packing of hydrophobic residues dispersed over the main part of the dimer interface is highly conserved among known a-glucan phosphorylases [22] including EcMalP and, by sequence similarity, CcStP It suggests that interfacial contacts mediated by polar groups must be different in EcMalP and CcStP We were interested to examine the relative timing of steps involved in dimer formation and the appearance of oxyanion-dependent stabilization of activity during reconstitution of apo-CcStP Analysis of time courses of recovery of enzyme activity in the absence and presence of phosphate showed that the yield but not the rate at which the activity was regained was strongly dependent on the added phosphate These observations are novel and consistent with a mechanism in which the active dimer is formed first, and enzyme–oxyanion interactions that are lacking in the monomer are utilized to shift the equilibrium towards the catalytically competent enzyme (Scheme 1) The data are in excellent agreement with the proposed pathway of thermal denaturation of CcStP [17] and contribute to an improved understanding of the effect of phosphate binding on the dimer stability of CcStP The evidence presented here and summarized in Scheme significantly advances the mechanism underlying oxyanion-dependent dimer stabilization because it was possible for the first time to investigate the properties of the native-like folded apo-monomer of CcStP Because of its low conformational stability under conditions of thermally induced dissociation of the CcStP subunits, the apo-monomer usually escaped detection in the previous studies of CcStP stability [17,18] Scheme Formation of active dimers of CcStP during reconstitution of the apo-phosphorylase with PLP in the absence and presence of phosphate M is the native-like folded monomer; M¢ is an irreversibly denatured monomer; D is the PLP-containing, active dimer; D* is the stabilized dimer bound to phosphate; Maggr is aggregated protein All monomeric forms lack enzyme activity The denaturation of D as shown is supported by evidence published elsewhere [17] Reconstitution of mutant apo-enzymes yielded results that were fully consistent with recent comparisons of thermoinactivation rates of the same mutants [15,18] After correction for differences in protein concentration used, the level of activity recovered during the burst phase was similar among wild-type and all mutants when no phosphate was present Therefore, this implies that the mutations did not cause changes in the association rate of the phosphorylase subunits Altered kinetic stabilities of the mutants, compared to wild-type, are therefore likely due to changes in protomer dissociation rate The effect of phosphate on the recovery of activity was sensitive to mutations in the dimer contact region R234A had lost much of the apparent affinity of the wild-type for phosphate, and a phosphate effect on activity recovery was lacking completely in R242A under the conditions used The data reinforce the conception [15] that the side chains of Arg234 and Arg242 have key roles in the mechanism by which phosphate binding induces a kinetically stabilized conformation of CcStP (Scheme 1) Restoration of enzyme activity in PL-reconstituted phosphorylase by exogenous phosphate The characterization of CcStP reconstituted with PL in place of the natural cofactor PLP yielded results that are relevant in the context of function of the 5¢-phosphate group in phosphorylase catalysis [1], as follows A number of studies using PL-RmGP have shown that the otherwise inactive PL-phosphorylase recovered up to 19% of wildtype activity when exogenous oxyanions were present Among a series of compounds tested phosphite was the most powerful activator anion of PL-RmGP [23,24] Using PL-CcStP, phosphate was  4.5-fold more effective than phosphite, and in saturating concentrations of mM it restored 60% of the original enzyme activity at pH 7.5 The data suggest that phosphate binds to the cleft vacated in PLCcStP through removal of the original cofactor 5¢-phosphate group; and the positions of the dissociable phosphate in PL-CcStP and the covalently bound phosphate in the native enzyme are probably similar 3328 R Griessler et al (Eur J Biochem 271) The direct comparison of pH profiles for the catalytic rates of CcStP and the complex PL-phosphorylase and phosphate can arguably provide mechanistic information because enzyme systems were analyzed whose active sites differed only by a minimal modification However, any interpretation must be tempered considering that in RmGP, slightly different binding modes for cofactorbound and mobile phosphate groups have been detected by X-ray crystallography [25] The question of interest was whether differences in pKa values for covalent and noncovalent phosphate (pKa ¼ 7.2 [23]) groups are mirrored in the corresponding pH-rate profiles The pKa values of the cofactor phosphate in unliganded EcMalP and the EcMalP–arsenate complex are 5.6 [26] and 6.7 [27], respectively The pKa of the 5¢-phosphate group in a model Schiff base is 6.2 [26] The available evidence for EcMalP defines a range of plausible pKa values for CcStP because residues interacting with the 5¢-phosphate group in EcMalP are completely conserved in CcStP Log ksyn for the wild-type decreased above an apparent pKa of 6.9 whereas a pKa value of 7.9 was calculated from the pH profile of log ksyn for PL-CcStP bound to phosphate Unfortunately, the activity of PLCcStP in the presence of activator phosphite was too low to permit determination of a reliable pH profile The observed DpKa of  1.0 pH units would agree reasonably with DpKa ¼ 1.2 predicted on the basis of pKa values of phosphate and the cofactor 5¢-phosphate in a model compound These data are consistent with a pH-dependent mechanism in which the cofactor phosphate must be protonated so that catalysis to a-glucan synthesis occurs [1,28,29] The pH profiles of log kpho for wild-type and PL-CcStP decreased above an apparent pKa value of  7.3 It is not possible to assign this pKa value to the pH-dependent ionization of a group on the reactive enzyme–substrate complex; obviously it could reflect the ionization of the substrate phosphate Acknowledgements The financial support from the Austrian Science Funds (P15118 and P11898 to B.N.) is gratefully acknowledged We thank Dr Dieter Palm for communicating a protocol for the preparation of apo-EcMalP References Palm, D., Klein, H.W., Schinzel, R., Buehner, M & Helmreich, E.J.M (1990) The role of pyridoxal 5¢-phosphate in glycogen phosphorylase catalysis Biochemistry 29, 1099–1107 Graves, D.J & Wang, J.H (1972) a-Glucan phosphorylases – chemical and physical basis of catalysis and regulation Ann Rev Biochem 7, 435–482 Feldmann, K., Zeisel, H.J & Helmreich, E.J.M (1976) Complementation of subunits from glycogen phosphorylases of frog and rabbit skeletal muscle and rabbit liver Eur J Biochem 65, 285–291 Tu, J.-I & Graves, D.J (1973) Association-dissociation properties of sodium borohydride-reduced phosphorylase b J Biol Chem 248, 4617–4622 Feldmann, K., Zeisel, H & Helmreich, E (1972) Interactions between native and chemically modified subunits of matrix-bound glycogen phosphorylase Proc Natl Acad Sci USA 69, 2278– 2282 Ó FEBS 2004 Shaltiel, S., Hedrick, J.L & Fischer, E.H (1966) On the role of pyridoxal 5¢-phosphate in phosphorylase II Resolution of rabbit muscle phosphorylase Biochemistry 5, 2108–2116 Hedrick, J.L., Shaltiel, S & Fischer, E.H (1969) Conformational changes and the mechanism of resolution of glycogen phosphorylase b J Biol Chem 8, 2422–2429 Pan, P., Schinzel, R., Palm, D & Christen, P (1993) Reaction of imidazole-citrate-deformed glycogen phosphorylase with amino acids Eur J Biochem 215, 761–766 Pfeuffer, T., Ehrlich, J & Helmreich, E (1972) Role of pyridoxal 5¢-phosphate in glycogen phosphorylase II Mode of binding of pyridoxal 5¢-phosphate and analogs of pyridoxal 5¢-phosphate to apophosphorylase b and the aggregation state of reconstituted phosphorylase proteins Biochemistry 11, 2136–2145 10 Shaltiel, S., Hedrick, J.L., Pocker, A & Fischer, E.H (1969) Reconstitution of apophosphorylase with pyridoxal 5¢-phosphate analogs Biochemistry 8, 5189–5196 11 Shimomura, S., Emman, K & Fukui, T (1980) The role of pyridoxal 5¢-phosphate in plant phosphorylase J Biochem 87, 1043– 1052 12 Tagaya, M., Shimomura, S., Nakano, K & Fukui, T (1982) A monomeric intermediate in the reconstitution of potato apophosphorylase with pyridoxal 5¢-phosphate J Biochem 91, 589–597 13 Watson, K.A., Schinzel, R., Palm, D & Johnson, L.N (1997) The crystal structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase EMBO J 16, 1–14 14 Hudson, J.W., Golding, G.B & Crerar, M.M (1993) Evolution of allosteric control in glycogen phosphorylase J Mol Biol 234, 700–721 15 Griessler, R., Schwarz, A., Mucha, J & Nidetzky, B (2003) Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae Eur J Biochem 270, 2126–2136 16 Weinhausel, A., Griessler, R., Krebs, A., Zipper, P., Haltrich, D., ă Kulbe, K.D & Nidetzky, B (1997) a-1,4-D-glucan phosphorylase of gram-positive Corynebacterium callunae: isolation, biochemical properties and molecular shape of the enzyme from solution X-ray scattering Biochem J 326, 773–783 17 Griessler, R., D’Auria, S., Tanfani, F & Nidetzky, B (2000) Thermal denaturation mechanism of starch phosphorylase from Corynebacterium callunae: oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein Protein Sci 9, 1149–1161 18 Nidetzky, B., Griessler, R., Pierfederici, F., Psik, B., Scire, A & Tanfani, F (2003) Mutagenesis of the dimer interface region of Corynebacterium callunae starch phosphorylase alters the oxyanion ligand-dependent conformational relay that enhances oligomeric stability of the enzyme J Biochem (Tokyo) 134, 599–606 19 Eis, C., Griessler, R., Maier, M., Weinhausel, A., Bock, B., Hală ă trich, D., Kulbe, K.D., Schinzel, R & Nidetzky, B (1997) Efficient downstream processing of maltodextrin phosphorylase from Escherichia coli and stabilization of the enzyme by immobilization onto hydroxyapatite J Biotechnol 58, 156–166 20 Oikonomakos, N.G., Acharya, K.R., Stuart, D.I., Melpidou, A.E., McLaughlin, P.J & Johnson, L.N (1988) Uridine (5¢)diphospho(1)-a-D-glucose A binding study to glycogen phosphorylase b in the crystal Eur J Biochem 173, 569–578 21 Holm, L & Sander, C (1995) Evolutionary link between glycogen phosphorylase and a DNA modifying enzyme EMBO J 14, 1287–1293 22 Lin, K., Hwang, P.K & Fletterick, R.J (1997) Distinct phosphorylation signals converge at the catalytic center in glycogen phosphorylases Structure 5, 1511–1523 23 Chang, Y.C., McCalmont, T & Graves, D.J (1983) Functions of the 5¢-phosphoryl group of pyridoxal 5¢-phosphate in phos- Ĩ FEBS 2004 24 25 26 27 Cofactor dissociation studies of starch phosphorylase (Eur J Biochem 271) 3329 phorylase: a study using pyridoxal-reconstituted enzyme as a model system Biochemistry 22, 4987–4993 Parrish, R.F., Uhing, R.J & Graves, D.J (1977) Effect of phosphate analogues on the activity of pyridoxal reconstituted glycogen phosphorylase Biochemistry 16, 4824–4831 Oikonomakos, N.G., Zographos, S.E., Tsitsanou, K.E., Johnson, L.N & Acharya, K.R (1996) Activator anion binding site in pyridoxal phosphorylase b: The binding of phosphite, phosphate, and fluorophosphate in the crystal Protein Sci 5, 2416–2428 Schinzel, R., Palm, D & Schnackerz, K.D (1992) Pyridoxal 5¢-phosphate as a 31P reporter observing functional changes in the active site of Escherichia coli maltodextrin phosphorylase after site-directed mutagenesis Biochemistry 31, 4128–4133 Becker, S., Schnackerz, K.D & Schinzel, R (1994) A study of binary complexes of Escherichia coli maltodextrin phosphorylase: a-D-glucose 1-methylenephosphonate as a probe of pyridoxal 5¢-phosphate–substrate interactions Biochim Biophys Acta 1243, 381–385 28 Watson, K.A., McCleverty, C., Geremia, S., Cottaz, S., Driguez, H & Johnson, L.N (1999) Phosphorylase recognition and phosphorolysis of its oligosaccharide substrate: answers to a long outstanding question EMBO J 18, 4619–4632 29 Geremia, S., Campagnolo, M., Schinzel, R & Johnson, L.N (2002) Enzymatic catalysis in crystals of Escherichia coli maltodextrin phosphorylase J Mol Biol 322, 413–423 Supplementary material The following material is available from http://blackwell publishing.com/products/journals/suppmat/EJB/EJB4265/ EJB4265sm.htm Fig S1 Column sizing experiment using Superose 12 HR 10/30 to determine the subunit association state of CcStP, apo-CcStP in the absence and presence of mM UDP-a-Dglucose, and the reconstituted enzyme  ... catalytic activity and stability We chose starch phosphorylase from Corynebacterium callunae (CcStP), which has been characterized biochemically and structurally [15,16], for particular reason... concentration of between 0.0 and 100 lM was added to reconstitute the holo-enzyme The reaction was carried out at 30 °C and typically, the time Ó FEBS 2004 Cofactor dissociation studies of starch phosphorylase. .. detect formation of possible hybrid dimers of CcStP and maltodextrin phosphorylase from Escherichia coli (EcMalP) Finally, we show results from kinetic studies of CcStP reconstituted with pyridoxal