This proposal outlines an experimental approach to understanding the detailed chemical mechanisms of the bacterial enzyme tartrate dehydrogenase (TDH). TDH is an unusual enzyme in that it recognizes three different substrates and catalyzes a different chemical reaction with each one. The rate-determining steps in the catalytic reactions will be determined by steady-state kinetic studies, intermediate-partitioning experiments, and isotope effect studies. Experiments are described which will allow calculation of the intrinsic isotope effects on the enzymatic reactions, from which the transition state structures, and thus the origin of the catalytic power, can be inferred. Substrate analog studies will probe the molecular determinants of each reaction, and the energetic costs of following one reaction pathway versus another. The gene encoding TDH will be cloned, and randomly mutagenized and expressed to determine whether a protein can be created which has enhanced ability to catalyze the slowest reaction catalyzed by the wild-type enzyme. The mutant proteins will be characterized kinetically to determine the basis for their increased efficiency, and to determine whether the ability to catalyze the other reactions has changed. Such studies should identify which segments of an enzymatic reaction are most easily optimized, and the roles that various enzyme-substrate interactions play in determining the outcome of the catalytic reaction. Understanding the mechanisms by which the rates of enzymatic reactions are limited, and the mechanisms by which substrate reactivity is controlled, is necessary if enzymes are to be manipulated in a rational manner for use in biotechnology and understood as targets for efficient design of pharmaceuticals. TDH is an ideal system for such studies because it appears to be finely balanced as a catalyst for three different reactions, so that subtle changes in its interactions with the substrates have a dramatic effect on the outcome of the catalytic reaction.
| Tipton, P A; Beecher, B S (1994) Tartrate dehydrogenase, a new member of the family of metal-dependent decarboxylating R-hydroxyacid dehydrogenases. Arch Biochem Biophys 313:15-21 |
| Beecher, B S; Koder, R L; Tipton, P A (1994) Tartrate dehydrogenase-oxalate complexes: formation of a stable analog of a reaction intermediate complex. Arch Biochem Biophys 315:255-61 |
| Tipton, P A (1993) Intermediate partitioning in the tartrate dehydrogenase-catalyzed oxidative decarboxylation of D-malate. Biochemistry 32:2822-7 |
