Enzymatic transition state structure can be experimentally approached by a combination of intrinsic kinetic isotope effects (KIEs) and computational quantum chemistry. Experimental KIEs provide boundaries for computational transition states. Transition state knowledge provides chemical insights and practical application of molecular electrostatic maps of transition states are blueprints for transition state analog design. Transition state analysis has provided femtomolar to picomolar inhibitors for N-ribosyltransferases, some of the most powerful enzyme inhibitors. Several transition state analogs are in human clinical trials with more in preclinical studies. These analogs follow the predictions of Pauling and Wolfenden that transition state mimics bind tightly by engaging the forces permitting enzymes to accelerate reactions up to 20 orders of magnitude. Yet, the application of transition state structure to inhibitor design is in its infancy, with development fr only a few chemical reaction classes. The fundamental processes leading to transition state formation remain in contention. This project will expand the utility of transition state analysis ad inhibitor design to two new reaction classes. A fundamental property of enzymatic transition state formation will be tested by individual heavy amino acid substitution into catalytic sites.
Th first aim will target human phenylethanolamine N- methyltransferase (PNMT), the SAM-based formation of epinephrine. N-Methylation reactions are critical for hormones and neurotransmitter metabolism. PNMT is a target for blood pressure regulation and has potential links to Alzheimer's disorder. Hydrolysis of beta-lactamases is our second target class. New Delhi zinc metallo-beta-lactamase (NDM-1) and serine-beta- lactamase are clinically important targets for antibiotic resistance and provide new transition state insight for beta-lactam hydrolysis. The thir aim develops a new experimental approach to resolve fast (fs-ps) protein motions linked to transition state barrier-crossing. Pioneering enzyme chemistry experiments have revealed that increased protein mass slows on-enzyme chemistry. The ultimate extension of heavy-enzyme technology will replace selected amino acids specifically in the catalytic site with their heavy (2, 13C, 15N) counterparts by cell-free protein synthesis with a single amino acid substitution. This project advances transition state chemistry, transition state analog design and the fundamental properties of enzymatic catalysis.

Public Health Relevance

The National Institutes of Health, the World Health Organization and the Center for Disease Control are concerned that the development of new drugs is lagging behind the rise of drug resistance and the evolution of new pathogens. Drug design by transition state analysis provides an efficient approach to drug design. However, the field is limited to only a few classes of enzymatic transition states. This research expands the fundamental knowledge of enzymatic transition states to two new classes of enzyme chemistry. They are pertinent to central nervous system disorders and antibiotic resistance. Drug design from transition state structure is still developing. However, several candidates have reached clinical trials, providing proof of concept for this method.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Barski, Oleg
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Albert Einstein College of Medicine, Inc
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Harijan, Rajesh K; Zoi, Ioanna; Antoniou, Dimitri et al. (2018) Inverse enzyme isotope effects in human purine nucleoside phosphorylase with heavy asparagine labels. Proc Natl Acad Sci U S A 115:E6209-E6216
Evans, Gary B; Tyler, Peter C; Schramm, Vern L (2018) Immucillins in Infectious Diseases. ACS Infect Dis 4:107-117
Ducati, Rodrigo G; Namanja-Magliano, Hilda A; Harijan, Rajesh K et al. (2018) Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum. Proc Natl Acad Sci U S A 115:2114-2119
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Ducati, Rodrigo G; Firestone, Ross S; Schramm, Vern L (2017) Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum. Biochemistry 56:6368-6376
Namanja-Magliano, Hilda A; Stratton, Christopher F; Schramm, Vern L (2016) Transition State Structure and Inhibition of Rv0091, a 5'-Deoxyadenosine/5'-methylthioadenosine Nucleosidase from Mycobacterium tuberculosis. ACS Chem Biol 11:1669-76
Du, Quan; Wang, Zhen; Schramm, Vern L (2016) Human DNMT1 transition state structure. Proc Natl Acad Sci U S A 113:2916-21

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