[...]... kinds of enzymes that can be investigated and the hypotheses that can be tested Therefore, the elucidation of an enzyme mechanism requires the steady-state data and the hypotheses confirmed or eliminated along with data and hypotheses from other techniques such as x-ray crystallography, fast-reaction kinetics, and others Certain kinds of enzymes are difficult to study Polymerase and depolymerase enzymes... steady-state enzyme kinetics Finally a generally useful sequence of experiments in an investigation of steady-state enzyme kinetics will be described 1.2 What is the Steady-State? In a prototypical experiment an enzyme reaction is initiated by the combination of free enzyme and substrate rather instantly compared to the other things that happen, whether this is accomplished by the simple addition of enzyme. .. best As will be seen in the remainder of the book the marriage of steady-state enzyme kinetics and mathematical modeling has been a productive and happy one One of the reasons for the rather happy marriage between steady-state enzyme kinetics and mathematical modeling is that the investigator is frequently faced with data and only a limited set of reasonable mathematical models that are also related... are stand alone programs, but usually include some graphics; and specific curvefitting programs for enzyme kinetics The large statistical packages such as Matlab® and MLAB® (Table 3.1) are very versatile but expensive and usually require the operator to write some program steps to call the routines The second category, the stand-alone general curvefitting programs such as Scientist®, Fitall®, and CurveExpert... concentration comparable to that of the enzyme in order to measure a significant reaction rate Therefore, the polymer cannot be treated as a substrate for steady-state modeling purposes, and investigations with these enzymes are limited In addition it is possible to test hypotheses about the number and kinds of intermediates and transition states between the binding of substrate and the release of product only... initial velocity of the reaction These phenomena can be illustrated in a simulated reaction (Figure 1.1) in which the free enzyme (E) forms an enzyme- substrate intermediate (EA) and the latter subsequently forms an enzyme- product intermediate (EP), which dissociates to product (P) and free enzyme It can be seen that the product concentration endures a short lag period, while the concentration of both intermediates... evaluated with the first estimates of and ( and the second term is the product of the first derivative of the Michaelis-Menten equation with respect to and the difference the first estimate and the second estimate of KM ; the third term is the product of the first derivative of the Michaelis-Menten equation with respect to Vmax and the difference of the first estimate and the second estimate of Since... STEADY-STATE KINETICS 1.1 Introduction This book contains a number of concepts and agendas implicit in steady-state enzyme kinetics that will hopefully be made explicit in this chapter Specifically this chapter will contain a description of the steady state as well as the assumptions and approximations associated with it It will contain a general description of the things that can be learned from and the... the value of y equal to zero, estimate the best value for and solve for the difference between and Find the value of and start the iteration over Repeat the process until the differences become very small This method was first applied to the Michaelis-Menten equation by Wilkinson [3], and a more systematic explanation of it is provided by Cleland [5] Application to the Michaelis-Menten equation looks... conditions The development of the models and their testing with data is known as mathematical modeling The present chapter describes the use of mathematical modeling in testing models for data from investigations of steady-state enzyme kinetics The purpose here is for the reader to achieve an understanding of the process as well as the assumptions involved and to be comfortable with the software available .