Abstract
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 Sn, n = 1–4 and S’n, 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 (1H 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. The emergence of short hydrogen bonds at the binding interface of effectors and thrombin at remote exosites has recently gained recognition. Herein, I describe our contribution, a confirmation of this discovery, by low-field 1H NMR. The principal conclusion of this review is that proton sharing at distances below the sum of van der Waals radii of the hydrogen and both donor and acceptor atoms contribute to the remarkable catalytic prowess of serine proteases of the blood clotting system and other enzymes that employ acid-base catalysis. Proton bridges also play a role in tight binding in proteins and at exosites, i.e., allosteric sites, of enzymes.
Highlights
Fundamental questions of the origins and evolutionary progress of catalytic acceleration by enzymes intrigued and motivated enzymologists for some time
Our work extended to several serine proteases and cholinesterases, which revealed a dependence of the degree of participation of proton bridges in the stabilization of the transition state (TS) of substrate reactions on sub-site interactions in the specificity pocket [20,21,22,23,24,25,26]
From the low-field signal intensity measured in different isotopic mixtures of buffered water, an isotope effect of 2.2 ± 0.2 was calculated for the formation of a proton bridge which occurs at the active site in the PPACK-inhibited thrombin
Summary
Fundamental questions of the origins and evolutionary progress of catalytic acceleration by enzymes intrigued and motivated enzymologists for some time. The hirunorms have three amino acids of the N-terminal sequence of hirudin followed by a linker and 10 amino acids of the C-terminus of hirudin, but are about hundred times less effective inhibitors of α-thrombin than the parent compound One novelty in this endeavor has been the characterization of short proton bridges at binding sites near the active-site cleft and remote sites by full and partial KSIE probes and high-resolution 1H NMR techniques [20,21,22,23,26]. These interactions have important roles in binding of the extended substrates and inhibitors and enforcing the conformation required for efficient catalysis. Other works with substrates and inhibitors demonstrated that P’ sites [75,76,77,78] and exosites [26] in long peptide and protein substrates can have roles in exerting compression at the active site
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