Richard Wolfenden, PhD

• Wolfenden Lab Website
• Publication List

RESEARCH INTERESTS:

Transition State Affinity, Enzyme Catalysis and the Depth of Chemical Time

As one approach to studying enzyme reaction mechanisms, I have been fascinated by M. Polanyi’s early (1921) recognition that the action of a catalyst depends on its ability to bind the altered substrate in the transition state much more tightly than it binds the substrate in the ground state. That interest led to the design of stable “transition state analogues”, that have now been prepared against enzymes of every class and include prototypes of agents for treating hypertension and the spread of AIDS.  We use these inhibitors to investigate mechanisms, by determining the structures of their enzyme complexes using X-ray, NMR and mass spectrometry; and by modifying the enzyme or inhibitor and observing the consequences for binding and catalysis.  To evaluate the affinity that any enzyme develops for altered substrate in the transition state, it is necessary to establish the rate of the corresponding reaction in water in the absence of a catalyst.   By extrapolation of rate constants obtained at elevated temperatures, we find that the rates of biological reactions span a range of ~ 1019-fold, with some half-times exceeding the age of the Earth.  Such findings are helpful in identifying enzymes that are especially sensitive targets for drug design.

Levels of Binding Affinity Achieved by Known Enzymes

Adenosine and cytidine deaminases bind substrates and products in forms corresponding to those that are most abundant in solution, but we find that other molecules are bound by adenosine and cytidine deaminases as very rare covalent hydrates that serve as transition state analogue inhibitors, with affinities that exceed the apparent affinities of substrates by 8 orders of magnitude. In these inhibitory hydrates, replacement of a single hydroxyl group by hydrogen, reduces their binding affinity by ~10 kcal/mole. OMP decarboxylase, the most powerful catalyst we know that acts as a pure protein without metals or other cofactors, shows similar effects.  Analysis of the corresponding affinities of pieces of these inhibitors, and of mutant enzymes, indicates the presence of major entropic effects of substituents (or, alternatively, enzyme binding determinants), acting together. Considered in conjunction with changes in enzyme shape that accompany transition state analogue binding, these entropic effects help to explain the astonishingly large changes in binding affinity that accompany the substrate’s progress along the reaction coordinate.  There are indications that  these entropy changes may also include an important role for solvent water.

Solvent Water and Biochemical Recognition

We are using water-to-vapor distribution measurements to determine the absolute affinities or solvent water of the peptide bond, amino acid side chains, carbohydrate derivatives, nucleic acid bases and phosphoric acid derivatives. The free energy of peptide bond biosynthesis, or of the hydrolysis of ATP, for example, can be explained entirely in terms of changing free energy of solvation). Differences in the strength of solvation of different forms (cis v. trans) of the peptide bond, and of different amino acid side-chains, appear to be of vital importance in establishing the 3-dimensional folding of proteins. These affinities must also play a significant role in molecular recognition (including enzyme-substrate recognition in the ground and transition states), because complex formation involves the stripping of water from between the interacting partners, at the positions where they make contact. We are analyzing those effects, and also investigating the occasional circumstances under which water can enter or be "dragged" into nonpolar surroundings.

RECENT PUBLICATIONS:

Schroeder GK, Wolfenden R. Rates of spontaneous disintegration of DNA and the rate enhancements produced by DNA glycosylases and deaminases. Biochemistry. 2007 Nov 27;46(47):13638-47. Epub 2007

Lewis CA Jr, Wolfenden R. Indiscriminate binding by orotidine 5'-phosphate decarboxylase of uridine 5'-phosphate derivatives with bulky anionic c6 substituents.

Biochemistry. 2007 Nov 20;46(46):13331-43. Epub 2007

Wolfenden R. Experimental measures of amino acid hydrophobicity and the topology of transmembrane and globular proteins. J Gen Physiol. 2007 May;129(5):357-62. Epub 2007

Schroeder GK, Wolfenden R. The rate enhancement produced by the ribosome: an improved model. Biochemistry. 2007 Apr 3;46(13):4037-44. Epub 2007  

Wolfenden R, Yuan Y. Monoalkyl sulfates as alkylating agents in water, alkylsulfatase rate enhancements, and the "energy-rich" nature of sulfate half-esters. Proc Natl Acad Sci U S A. 2007 Jan 2;104(1):83-6. Epub 2006

Wolfenden R. Degrees of difficulty of water-consuming reactions in the absence of enzymes. Chem Rev. 2006 Aug;106(8):3379-96

Huang DT, Kaplan J, Menz RI, Katis VL, Wake RG, Zhao F, Wolfenden R, Christopherson RI. Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction.

Biochemistry. 2006 Jul 11;45(27):8275-83

Callahan BP, Lomino JV, Wolfenden R.Nanomolar inhibition of the enterobactin biosynthesis enzyme, EntE: synthesis, substituent effects, and additivity. Bioorg Med Chem Lett. 2006 Jul 15;16(14):3802-5. Epub 2006

Schroeder GK, Lad C, Wyman P, Williams NH, Wolfenden R. The time required for water attack at the phosphorus atom of simple phosphodiesters and of DNA. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4052-5. Epub 2006

Callahan BP, Bell AF, Tonge PJ, Wolfenden R. A Raman-active competitive inhibitor of OMP decarboxylase. Bioorg Chem. 2006 Apr;34(2):59-65. Epub 2006