Binding modes for substrate and a proposed transition-state analogue of protozoan nucleoside hydrolase.
The transition-state structure for inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase) from Crithidia fasciculata is characterized by oxycarbonium character in the ribosyl and weak bonds to the departing hypoxanthine and incipient water nucleophile [Horenstein, B. A., Parkin, D. W., Estupiñán, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. Inhibitors designed to resemble the transition state are slow-onset, tight-binding inhibitors with observed Km/Ki values up to 2 x 10(5) [Schramm, V. L., Horenstein, B. H., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262]. Although slow-onset, tight binding is consistent with transition-state stabilization, more direct evidence can be obtained by comparing the groups which interact with the substrate to provide binding and catalysis with those which interact with the putative transition-state inhibitor. The Km value for inosine binding to IU-nucleoside hydrolase is independent of pH over the range 5.6-10.5. Dependencies of Vmax and Vmax/Km on pH result in pH optima near 8.0. A single group with pK of 9.1 must be protonated for catalytic activity, and protonation of a second group with a pK of 7.1 results in loss of activity. 1-(S)-Phenyl-1,4-dideoxy-1,4-imino-D-ribitol (phenyliminoribitol) binds with an equilibrium Kd of 30 nM and has been proposed to be a transition-state inhibitor. The pH dependence for the competitive inhibition by phenyliminoribitol resembles the Vmax profile with the protonation of a single group, pK 7.5, required for inhibitor binding and the protonation of a subsequent group, pK 6.6, causing loss of binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Parkin
D W
DW
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Schramm
V L
VL
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D016831:Crithidia fasciculata / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007288:Inosine / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D012255:Ribitol / Q000031:analogs & derivatives / Q000494:pharmacology; D014529:Uridine / Q000378:metabolism*
D004791:Enzyme Inhibitors; C089322:phenyliminoribitol; D012255:Ribitol; D007288:Inosine; D009699:N-Glycosyl Hydrolases; C096466:inosine-uridine hydrolase; D014529:Uridine