The research described in this proposal aims to provide a deeper understanding of enzyme catalysis. We have three major goals. First, we shall study the mechanisms of action of several enzymes in the shikimate pathway. This metabolic sequence is responsible for the synthesis of the three aromatic amino acids in bacteria and in plants, and the enzymes in this pathway are potential targets for spectoscopic and fast quench techniques.
We aim first to deconvolute the mechanistic steps and the reaction intermediates of three enzymes: chorismate mutase(where we shall clone the small monofunctional enzyme from Bacillus and study the enzymes:substrate complex directly by 13 C NMR and FTIR, 3-deoxy-D- arabinoheptulsonic acid-7-phosphate synthase (where the role of metal ion, and the curious nature of the enzyme's handling of phosphoenolpyruvate will be probed), and dehydroquinate synthase (for which we shall synthesize a logical sequence of blocked substrate analogues and likely mechanism-based inactivators). Second, we shall continue our investigations of the catalytic devices that are used more generally by enzymes with especial reference to the internal thermodynamics of enzyme systems. Our goal is to develop and refine our understanding of the relationships between substrate binding free energy and catalytic effectiveness. We shall exploit two well-characterized metabolic pathways in this programme: glycolysis (the enzymes from which are readily available from mammalian muscle) and the shikimate pathway (all the enzymes of which have now been cloned behind strong promoters). Our focus on the enzymes from these sequences will allow explicit tests to be made of our theories on the optimization of catalytic efficiency in vivo. Third, we shall initiate a novel series of experiments that aims to trace the development of new catalytic function. We shall first concentrate on the development of catalytic capability in two systems. By random mutagenesis and strong selection, we shall go from the D-Ala,D-Ala carboxypeptidase/transpeptidase of Streptomyces to a beta- lactamase (the enzyme that is largely responsible for the resistance of bacterial populations to penicillin), and from the class II aldolase of E coli to a triosephosphate isomerase. In each of these cases, the starting enzyme is well-characterized and catalyzes half of the target reaction. Moreover, we have powerful selection available in the search for the new catalytic activities. This search for 'enzyme evolvants' will evaluate the possibility of generating new catalytic entities, and will test our ability to change both the specificity and the mechanistic versatility of enzyme catalysts.
| Kountz, P D; Freeman, S; Cook, A G et al. (1988) The stereochemical course of phospho group transfer catalyzed by rat liver 6-phosphofructo-2-kinase. J Biol Chem 263:16069-72 |
