John P. Richard

Richard has conducted parallel studies on the mechanisms for organic reactions in aqueous solution and at enzyme active sites in order to define the root causes for enzymatic rate accelerations. The focus of many of these studies has been on the characterization of the lifetimes and thermodynamic stability for carbocation and carbanion intermediates of organic reactions in water and the determination of the mechanisms for their stabilization by enzyme catalysts.^[12][13]

Richard's postdoctoral work described the use of an azide anion clock to determine the lifetimes of carbocation intermediates of solvolysis reactions.^[14] He showed that these lifetimes sometimes enforce the mechanisms for nucleophilic substitution at aliphatic carbon.^[15][16][17] Richard and Amyes next reported novel methods for determination of the pK_as of weak carbon acids in water,^[18] and their application in the determination of the effect of a spectrum of organic functional groups on carbon acid pK_a.^[19] His work has focused on creating a model to rationalize the large effects of resonance electron-donating or accepting substituents on the lifetimes of carbocation and carbanion intermediates of organic reactions.^[20][21]

Richard has worked to draw comparisons between the mechanisms for the formation of carbocations and carbanions in water and at enzyme active sites. His application of the azide ion clock to the characterization of the oxocarbocation intermediate of ß-galactosidase-catalyzed hydrolysis of lactose showed that the intermediate is stabilized by interactions with the protein catalyst.^[22] His comparison of the pKas for the weakly acidic C-6 hydrogen of uridine monophosphate in water and at the active site of orotidine 5'-monophosphate decarboxylate demonstrated that there is a large stabilization of the UMP carbanion reaction intermediate by interactions with the protein catalyst.^[23] This was one key result from studies to characterize the mechanism of action of an enzyme that operates at peak catalytic efficiency.^[24] His investigations on the glycolytic enzyme triosephosphate isomerase revealed the mechanism by which the catalyst operates to increase the driving force for proton transfer from the enzyme-bound carbon acid to the protein.^[25]

Richard's investigations on the nonenzymatic isomerization and elimination reactions of triosephosphates have shed light on the origin of cellular methylglyoxal, a toxic compound that is neutralized by the action of glyoxalase I and II.^[26] His work has led to the identification of novel nonenzymatic Claisen and aldol condensation reactions of pyridoxal cofactor analogs, and results from collaborative studies with Crugeiras and Rios provide a characterization of the kinetics and thermodynamics for proton transfer reactions at pyridoxal-amino acid adducts.^[27] In collaboration with Richard Nagorski, it was demonstrated that Zn²⁺ catalyzes aldose-ketose isomerization through competing proton and hydride transfer mechanisms. This finding was predicted because the two mechanisms are followed by enzymes such as triosephosphate isomerase (proton transfer) and xylose isomerase (hydride transfer).^[28] Alongside Janet Morrow, Richard investigated small molecule metal-ion catalysts of phosphate diester hydrolysis in work that characterized cooperativity in catalysis by binuclear complexes and demonstrated that these complexes achieve enzyme-like rate accelerations.^[29]

Richard and Amyes discovered that many enzyme-catalyzed reactions of phosphodianion truncated substrates are activated by phosphite dianion.^[30][31] These enzymes utilize binding energy of the substrate phosphodianion to drive a change in protein conformation that traps the substrate at an active-site cage;^[32] this is equivalent to the substrate-induced fits first described by Daniel Koshand.^[33] The activating substrate-driven enzyme conformational changes result in the differential binding of enzymatic ground and transition states that is a required property of the most proficient enzyme catalysts.^[34] This model has provided a simple rationalization for the activation of adenylate kinase-catalyzed phosphoryl group transfer from adenosine triphosphate to phosphite dianion by the substrate fragment adenosine,^[35] as well as for the activation of formate dehydrogenase-catalyzed hydride transfer from formate to nicotinamide riboside by the substrate fragment ADP.^[36] The latter finding confirmed a proposal by W. P. Jencks that evolution has produced cofactors composed of small reactive functionalities connected to larger nonreactive fragments that provide large intrinsic binding energies for stabilization of enzymatic transition states.^[37]

• 2003 – Walton Visitor Fellow, University College, Dublin, Ireland^[12]
• 2009 – Jacob Schoellkopf Medal, ACS Western New York Section^[4]
• 2014 – Fellow, American Chemical Society^[7]
• 2020 – MIRA Award, NIH^[6]

• Richard, J. P. (1993). Mechanism for the formation of methylglyoxal from triosephosphates. Biochemical Society Transactions, 21(2), 549–553.
• Iranzo, O., Kovalevsky, A. Y., Morrow, J. R., & Richard, J. P. (2003). Physical and kinetic analysis of the cooperative role of metal ions in catalysis of phosphodiester cleavage by a dinuclear Zn (II) complex. Journal of the American Chemical Society, 125(7), 1988–1993.
• Amyes, T. L., Diver, S. T., Richard, J. P., Rivas, F. M., & Toth, K. (2004). Formation and stability of N-heterocyclic carbenes in water: the carbon acid p K a of imidazolium cations in aqueous solution. Journal of the American Chemical Society, 126(13), 4366–4374.
• Richard, J. P. (2019). Protein flexibility and stiffness enable efficient enzymatic catalysis. Journal of the American Chemical Society, 141(8), 3320–3331.
• Richard, J. P. (2022). Enabling role of ligand-driven conformational changes in enzyme evolution. Biochemistry, 61(15), 1533–1542.