John P. Richard, University at Buffalo, Department of Chemistry, Buffalo, NY, 14260, USA

Substrate binding occludes water from the active sites of many enzymes. There is a correlation between the burden to enzymatic catalysis of deprotonation of carbon acids and the substrate immobilization at solvent-occluded active sites for ketosteroidisomerase (KSI—small burden, substrate pKa = 13), triosephosphateisomerase (TIM, substrate
pKa ≈ 18) and diaminopimelateepimerase (DAP epimerase, large burden, substrate pKa≈ 29) catalyzed reaction. KSI binds substrates at a surface cleft, TIM binds substrate at an exposed ‘cage’ formed by closure of flexible loops; and, DAP epimerase binds substrate in a tight cage formed by an ‘oyster-like’ clamping motion of protein domains. The mechanistic imperatives for catalysis of deprotonation of α-carbonyl carbon by triosephosphateisomerase (TIM) are discussed. There is a strong imperative to reduce the large thermodynamic barrier for deprotonation of carbon to form an enediolate reaction intermediate and a strong imperative for specificity in the expression of the intrinsic phosphodianion binding energy at the transition state for the enzyme-catalyzed reaction. Binding energies of 2 and 6 kcal/mol, respectively, have been determined for the formation of phosphitedianion complexes to TIM and to the transition state for TIM-catalyzed deprotonation of the truncated substrate glycolaldehyde (T. L. Amyes, J. P. Richard, Biochemistry 2007, 46, 5841). We propose that the phosphitedianion binding energy, which is specifically expressed at the transition state complex, is utilized to stabilize a rare catalytically active loop-closed form of TIM. The results of experiments to probe the role of the side chains of Ile172 and Leu232 in activating the loop-closed form of TIM for catalysis of substrate deprotonation are discussed. Evidence is presented that the hydrophobic side chain of Ile172 assists in activating TIM for catalysis of substrate deprotonation through an enhancement of the basicity of the car- boxylate side chain of Glu167. Our experiments link the two imperatives for TIM-catalyzed deprotonation of carbon by providing evidence that the phosphodianion binding energy is utilized to drive an enzyme conformational change, which results in a reduction in the thermodynamic barrier to deprotonation of the carbon acid substrate at TIM compared with the barrier for deprotonation in water. The effects of a P168A mutation on the kinetic parameters for the TIM-catalyzed reactions of whole and truncated substrates are discussed.

Biography

John P. Richard received his B.S. degree in Chemistry from The Ohio State University. He remained in Columbus to do a Ph.D. under the direction of Perry Frey. This was followed by postdoctoral work with Bill Jencks at Brandeis University and then at the Fox Chase Cancer Center working with Nobel Laureate Ernie Rose. Richard has edited 16 books and published 180 papers in the chemical and biochemical literature. He served for six years as the Secretary of the American Chemical Society Division of Biological Chemistry and has participated in various capacities in the organization of international conferences, including the 2006 Gordon Research Conference on Enzymes, Coenzymes and Metabolic Pathways, the 2010 Gordon Research Conference on Isotopes in the Biological and Chemical Sciences and the 2011 Winter Enzyme Mechanisms Conference. Richard was the Editor of Annual Reports on the Progress of Chemistry, Section B from 1996 – 2001, and since 2000 has served as Editor of Advances in Physical Organic Chemistry. He is a member of the Editorial Advisory Board of Biochemistry, Bioorganic Chemistry and The Journal of Physical Organic Chemistry. As an independent investigator, Richard has studied the mechanisms of a wide range of organic reactions. These include nucleophilic substitution and proton transfer reactions at carbon; and, catalysis of phosphate diester hydrolysis by metal ion complexes. Richard’s research has been extended to studies of the mechanisms for the stabilization of reactive intermediates at the active sites of enzymes and this has led, inevitably, to studies that define the role of flexible protein loops in stabilizing these intermediate. Richard's work has been funded continuously since 1988 by grants from the National Institutes of Health and he has received additional support from the National Science Foundation and the Petroleum Research Fund.