Proton Bridging in Catalysis by and Inhibition of Serine Proteases of the Blood Cascade System.

Inquiries into the participation of short hydrogen bonds in stabilizing transition states and intermediate states in the thrombin, factor Xa, plasmin and activated protein C-catalyzed reactions revealed that specific binding of effectors at S, n = 1-4 and S', n = 1-3 and at remote exosites elicit complex patterns of hydrogen bonding and involve water networks. The methods employed that yielded these discoveries include; (1) kinetics, especially partial or full kinetic deuterium solvent isotope effects with short cognate substrates and also with the natural substrates, (2) kinetic and structural probes, particularly low-field high-resolution nuclear magnetic resonance (H NMR), of mechanism-based inhibitors and substrate-mimic peptide inhibitors. Short hydrogen bonds form at the transition states of the catalytic reactions at the active site of the enzymes as they do with mechanism-based covalent inhibitors of thrombin.

Proton bridging in the interactions of thrombin with hirudin and its mimics.

Thrombin is the pivotal serine protease enzyme in the blood cascade system and thus a target of drug design for control of its activity. The most efficient nonphysiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging, and hydrophobic interactions with thrombin to achieve tight binding with K(i) values in the nano- to femtomolar range.

Proton bridging in the interactions of thrombin with small inhibitors.

Thrombin is the pivotal serine protease enzyme in the blood cascade system. Phe-Pro-Arg-chloromethylketone (PPACK), phosphate, and phosphonate ester inhibitors form a covalent bond with the active-site Ser of thrombin. PPACK, a mechanism-based inhibitor, and the phosphate/phosphonate esters form adducts that mimic intermediates formed in reactions catalyzed by thrombin.

Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with DYNAFIT.

Hydrolytic reactions of oligopeptide 4-nitroanilides catalyzed by human-alpha-thrombin, human activated protein C and human factor Xa were studied at pH 8.0-8.4 and 25.

Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions.

Kinetic solvent isotope effects (KSIEs) for the factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of factor Va (FVa) and 5.0 x 10(-5) M phospholipid vesicles are slightly inverse, 0.82-0.

Full and partial deuterium solvent isotope effect studies of alpha-thrombin-catalyzed reactions of natural substrates.

Proton inventory studies of the thrombin-catalyzed fibrinogen activation to fibrinopeptide A are most consistent with a two-proton bridge forming at the transition state probably between Ser195 OgammaH and His57 Nepsilon2 and His57 Ndelta1 and Asp102 COObeta- at the active site, with fractionation factors 0.66 +/- 0.03 under enzyme saturation with substrate and 0.

Proton inventory studies of alpha-thrombin-catalyzed reactions of substrates with selected P and P' sites.

Deuterium kinetic solvent isotope effects for the human alpha-thrombin-catalyzed hydrolysis of (1) substrates with selected P(1)-P(3) sites, Z-Pro-Arg-7-amido-4-methylcoumarin (7-AMC), N-t-Boc-Val-Pro-Arg-7-AMC, Bz-Phe-Val-Arg-4-nitroanilide (pNA), and H-D-Phe-L-Pip-Arg-pNA, are (DOD)k(cat) = (2.8-3.3) +/- 0.