W. Wallace Cleland
University of Wisconsin-Madison
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Methods in Enzymology | 1982
W. Wallace Cleland
Publisher Summary This chapter discusses the use of pH studies to determine chemical mechanisms of enzyme-catalyzed reactions. The pH profiles that are of the most value are of pK i for competitive inhibitors or for metal-ion activators, log ( V/K ) for one or slower, nonsticky substrates, and isotope effects on V/K for these same substrates. Profiles of pKi for competitive inhibitors will show the required state of protonation of the inhibitor or of groups on the enzyme for binding. The log (V/K) versus pH profile for a nonsticky substrate shows the correct pK values of groups necessary for both binding and catalysis. Those pK values not present in the pKi profiles are groups that act as acid-base catalysts during the reaction, or whose protonation state is important for the chemical reaction, but not for binding. Profiles of V/K for nonsticky substrates can also be used in temperature-variation and solvent-perturbation studies to determine the nature of the groups involved.
Methods in Enzymology | 1980
W. Wallace Cleland
Publisher Summary This chapter discusses that the classical methods for measuring isotope effects involve direct comparisons of the rates with deuterated and nondeuterated substrates, giving both V/K and V isotope effects and isotope discrimination experiments, for tritium or heavier atom isotope effects that yield only V/K isotope effects. It reviews a new and very sensitive method for measuring isotope effects by equilibrium perturbation. The method requires that the reaction be reversible enough so that an equilibrium can be established, there be a color change or some other monitorable change, such as circular dichroism (CD), optical rotation, fluorescence, electron spin resonance (EPR) during the reaction, and the labeled compound have a high degree of isotope substitution, but be capable of measuring the isotope effects of 1.005, when the colored molecule is involved in the perturbation and about 1.002 when it is not. It is a cheap and rapid method that has proved to be extremely useful for the study of enzymic reactions.
Methods in Enzymology | 1982
W. Wallace Cleland
Publisher Summary Isotope effects have been used for a long time to determine the structures of transition states for chemical reactions. Such analysis is made easier by the fact that the chemical reaction being looked at is often the sole rate-limiting step. There are two kinds of isotope effects: primary and secondary. A primary isotope effect results from substitution of a heavy atom in a position where a total bond cleavage (or bond formation) occurs. Conversely, secondary isotope effects result from isotopic substitution in a position where no bonds are made or broken, but there is a change in the strength of the bonding during the reaction (or to be precise, the bonding in the transition state differs from that in the reactant). Primary and secondary isotope effects are often treated as separate phenomena, because with deuterium the primary ones tend to be much larger than the secondary ones. There are different isotope effects on V and on V/K for each reactant, and it is not always easy to determine the intrinsic isotope effect on the bond-breaking step, which is the value that gives information on the structure of the transition state. This chapter reviews the status of techniques for determining intrinsic isotope effects and discusses the interpretation of these values in terms of transition-state structure.
Methods in Enzymology | 1982
W. Wallace Cleland
Publisher Summary This chapter describes the preparation and properties of Co(III)- and Cr(III)-nucleotide complexes, and how they can be used to determine enzymic specificity. Before describing the preparation of complexes, the chapter reviews their stereochemistry and the types of isomerization that occur. The consideration is limited to complexes between phosphate oxygens and metal ion, since all of the complexes to be described are of that type. There are two methods described for synthesis of complexes: heating method and titration method. In heating method, a solution 10 mM is heated each in nucleotide and metal ion at 80°C for various times. Titration procedure converts monodentate CrATP cleanly in at least 90% yield to bidentate CrATP. Cr(III)-[and in some cases, Co(Ill)-] nucleotide complexes are usually excellent inhibitors of enzymes using Mg-nucleotide complexes as substrates, since their activities as substrates are normally very low, and thus they are useful as dead-end inhibitors of enzymic reactions in kinetic studies of mechanism, or for inducing suitable conformation changes without rapid turnover.
Methods in Enzymology | 1982
W. Wallace Cleland
Publisher Summary When one defines the kinetic constants for a mechanism, one normally defines more constants than there are independently determinable parameters, and thus some redundancy exists among the defined constants. The relationships among the various kinetic constants that express this redundancy are normally expressed as equations relating the equilibrium constant (Keq) to the kinetic constants and are called Haldanes. This chapter presents an analysis of Haldane relationships. There are two types of Haldanes, thermodynamic and kinetic, and every mechanism has at least one of each. The thermodynamic Haldane is the product of equilibrium constants for each step in the mechanism. The Kinetic Haldane is the ratio of the apparent rate constants in forward and reverse directions when all substrate concentrations are very low. For random mechanisms, one can assume either order of addition, and thus one obtains a number of similar Haldanes. For Ping Pong mechanisms, each half-reaction has its own Haldanes, and the Haldanes for the overall mechanism arc the product of those for the half reactions.
Methods in Enzymology | 1982
Ronald E. Viola; W. Wallace Cleland
Publisher Summary When there are three substrates for an enzymic reaction, the number of possible initial velocity patterns is large, and interpretation of the patterns is not as straightforward as when only two substrates are involved. Several attempts have been made to classify the possible terreactant mechanisms and distinguish between them because of initial velocity patterns and the resulting replots. This chapter discusses the form of the rate equation, the mechanistic interpretation of the absence of various terms from the rate equation, and experimental methods for establishing the presence or absence of such terms. Methods for distinguishing between kinetic mechanisms that give identical rate equations are also discussed. These results are served as a guide for interpretation of initial velocity patterns for terreactant mechanisms.
Methods in Enzymology | 1982
W. Wallace Cleland
Publisher Summary This chapter discusses the determination of equilibrium isotope effects by the equilibrium perturbation method. The equilibrium perturbation method is a very sensitive way of measuring kinetic isotope effects in which one adds enzyme to a system at equilibrium, but with one substrate labeled, and the corresponding product unlabeled. Because of the isotope effect, the initial rates of conversion of labeled substrate to product and of unlabeled product to substrate are not equal, and thus the reaction is perturbed from its equilibrium position. After isotopic mixing occurs, however, the rates in both directions become equal, and the system returns to its original position. The size of the perturbation from equilibrium depends on the size of the isotope effect, and is used to determine a value originally called app α, but which in the new notation now being used can be expressed as app D . In the normal analysis, this parameter and a knowledge of the equilibrium isotope effect are then used to calculate isotope effects in forward, D (Eq.P.) f , and reverse directions, D (Eq.P.) r , the ratio of which is D Keq. While for many reactions of interest D Keq values are easily measured, for heavy atom isotope effects with 13 C, 15 N, or 18 O, the values are much closer to unity and harder to determine.
Methods in Enzymology | 1979
W. Wallace Cleland
Methods in Enzymology | 1979
W. Wallace Cleland
Methods in Enzymology | 1999
W. Wallace Cleland; Dexter B. Northrop