Allosteric Regulation in Monomeric Enzymes
Human glucokinase catalyzes the ATP-dependent phosphorylation of glucose in the first reaction of glycolysis. This chemical transformation is the rate-limiting step of glucose metabolism in the human liver and pancreas. As such, glucokinase is a central regulator of glucose homeostasis. Genetic lesions within the glk gene that impair function result in maturity-onset diabetes of the young (MODY), while mutations that enhance catalysis cause hyperinsulinemia of infancy. These two disease states demonstrate the importance of precisely regulating glucokinase activity in vivo and emphasize the potential of therapeutically manipulating this key metabolic enzyme. Indeed, small-molecule glucokinase activators that enhance catalysis by as little as 6-fold are potential diabetes therapeutic agents. Glucokinase is a monomeric enzyme with the unique ability to be allosterically regulated by its substrate, D-glucose. The steady-state velocity of glucose phosphorylation is not hyperbolic, but instead displays a sigmoidal response to increasing sugar concentrations. The mechanistic basis for this kinetic cooperativity is unknown. Several models of kinetic cooperativity in monomeric enzymes have been formulated, yet none have been experimentally proven for human glucokinase. The goal of this project is to elucidate the mechanistic basis for cooperativity in human glucokinase. We wish to provide a kinetic and molecular description of the slow conformational transitions that are believed to give rise to cooperativity. We also wish to understand the mechanism of activation afforded by individual mutations in the glk gene that lead to hyperinsulinemia.
Enzyme Functional Dynamics
Enzymes lower activation barriers for chemical reactions by binding the altered substrate in the transition state with high affinity. During catalysis enzymes discriminate between the substrate in the ground state and the altered substrate in the transition state by a factor equaling the enzymatic rate enhancement - a value that often exceeds 1010. Understanding the thermodynamic basis for high-affinity transition state binding is essential for elucidating the nature of enzyme catalysis. The goal of this project is to investigate whether changes in protein conformational entropy contribute to transition state discrimination, thus representing a mechanism by which alterations in protein dynamics facilitate catalysis. As a model system for our investigations, we use adenosine deaminase (ADA), a 40 kDa, monomeric enzyme that catalyzes the hydrolytic deamination of adenosine to form inosine. During the course of deamination ADA binds the transition state 1012-fold more tightly than the ground state. A substantial portion of this differential binding affinity is reflected in the dissociation constant of 2`-deoxycoformycin (KD≈10-13 M), a stable transition state analog containing a tetrahedral carbon that mimics the attack of a water molecule at the C-6 position of adenosine. We are using state-of-the-art NMR methods to investigate side chain methyl group dynamics in the absence and presence of a ground state analog and 2’-deoxycoformycin to quantify changes in protein conformational entropy along the reaction coordinate. NMR data will be evaluated alongside the results of kinetic and calorimetric measurements aimed at establishing the enthalpic and entropic basis of ground state recognition and transition state discrimination by ADA. The results will establish the extent to which changes in protein conformational entropy contribute to enzyme catalysis.
Processes such as the acquisition of antibiotic resistance by pathogenic bacteria and the bioremediation of anthropogenic toxins by microorganisms clearly demonstrate that the evolution of new biological catalysts is an ongoing process in Nature. What is the origin of these new activities, and how do they evolve to yield highly specialized catalysts? The answers to these questions are not well understood, especially at the molecular level. The goal of this project is to combine microbial genetic selection strategies with mechanistic enzymology to identify new enzymes and investigate how their functions evolve. Once new enzymes are identified, we aim to use directed evolution to optimize the catalytic activities of latent enzymes in order to study the evolutionary processes at the molecular level. One potential source of new enzyme activities is promiscuous catalysts – enzymes that possess the ability to catalyze multiple chemical reactions that often share a common mechanistic step. Unfortunately, few examples of naturally promiscuous enzymes have been discovered, and little is known about the evolutionary processes that lead to the refinement of these rudimentary catalysts. Our laboratory has developed an experimental strategy to enable the rapid identification of functionally promiscuous enzymes. To date we have employed this strategy to identify new enzyme activities and previously undescribed metabolic pathways that are embedded within contemporary proteomes. The results of these studies reveal an unprecedented level of catalytic potential hidden inside microbial genomes and should provide valuable insight into how promiscuous progenitor catalysts are recruited to provide new functions.