The goals of this research project are to characterize the transition states of biologically significant enzymes of N-ribosyl bond scission. The transition states are used as atomic templates to develop the theory and practice of transition state inhibitor design. Transition state information and transition state analogies are used to investigate the nature of enzyme-bound transition states. The enzymes of interest include the protozoan nucleoside N-ribohydrolase isozymes and purine nucleoside phosphorylase. Protozoan parasites are purine auxotrophs and use three isozyme of N-ribohydrolases for purine salvage. The enzymes are not found in mammals. Purine nucleoside phosphorylase is widely distributed and is essential for normal T-cell function. Transition state structures for these enzymes have bee determined by kinetic isotope effect measurements using normal mode semiemperical and ab initio structural analysis. The first generation of transition state inhibitors have been synthesized and will be characterized. Experimental approaches will include: 1) laser and infrared spectroscopic investigation of the bound calcium-water center in the nucleoside hydrolases; NMR and Raman spectra of bound transition state inhibitors using isotope-edite difference analysis; and free-electron induced infrared lasers to study the actual enzymatic transition state; 2) x-ray crystallography of complexes with substrate and transition state analogues to define changes in protein structur in empty enzyme, Michaelis and transition state complexes; 3) development of theory for predicting binding energies of transition state inhibitors using trained pattern recognition; synthesis and testing of likely transition state inhibitors. The most powerful inhibitors will be tested for function in mice. The results of these studies are expected to give novel information about the nature of enzymatic transition states, provide new theory for the design of transition state inhibitors, and to provide powerful transition state inhibitors for several clinically relevant disorders.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Biochemistry Study Section (BIO)
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Ikeda, Richard A
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Albert Einstein College of Medicine
Schools of Medicine
United States
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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
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
Mason, Jennifer M; Yuan, Hongling; Evans, Gary B et al. (2017) Oligonucleotide transition state analogues of saporin L3. Eur J Med Chem 127:793-809
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; Evans, Gary B; Harijan, Rajesh K et al. (2017) Transition State Analogue Inhibitors of 5'-Deoxyadenosine/5'-Methylthioadenosine Nucleosidase from Mycobacterium tuberculosis. Biochemistry 56:5090-5098
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
Yuan, Hongling; Stratton, Christopher F; Schramm, Vern L (2016) Transition State Structure of RNA Depurination by Saporin L3. ACS Chem Biol 11:1383-90
Wang, Shanzhi; Cameron, Scott A; Clinch, Keith et al. (2015) New Antibiotic Candidates against Helicobacter pylori. J Am Chem Soc 137:14275-80
Suarez, Javier; Schramm, Vern L (2015) Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase. Proc Natl Acad Sci U S A 112:11247-51

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