Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transition-state inhibitor

Structurally diverse organophosphonate inhibitors targeting the active site of the enzyme were used to investigate the relationship of the active site and the dimer interface of wild-type protease in solution. Positional scanning synthetic combinatorial libraries revealed Kaposi's sarcoma-associated herpesvirus protease to be highly specific, even at sites distal to the peptide bond undergoing hydrolysis. Specificity results were used to synthesize a hexapeptide diphenylphosphonate inhibitor of Kaposi's sarcoma-associated herpesvirus protease. The transition state analog inhibitors covalently phosphonylate the active site serine, freezing the enzyme structure during catalysis. An NMR-based assay was developed to monitor the native monomer-dimer equilibrium in solution and was used to demonstrate the effect of protease inhibition on the quaternary structure of the enzyme. NMR, circular dichroism, and size exclusion chromatography analysis showed that active site inhibition strongly regulates the binding affinity of the monomer-dimer equilibrium at the spatially separate dimer interface of the protease, shifting the equilibrium to the dimeric form of the enzyme. Furthermore, inhibitor studies revealed that the catalytic cycles of the spatially separate active sites are independent. These results (i) provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, (ii) establish a molecular mechanism of protease activation and stabilization during catalysis, and (iii) highlight potential implications of substoichiometric inhibition of the viral protease in developing herpesviral therapeutics

The quest for accurate theoretical models of metalloenzymes: An aid to experiment

Enzymes are versatile oxidants in Nature that catalyze a range of reactions very efficiently. Experimental studies on the mechanism of enzymes are sometimes difficult due to the short lifetime of catalytic cycle intermediates. Theoretical modeling can assist and guide experiment and elucidate mechanisms for fast reaction pathways. Two key computational approaches are in the literature, namely quantum mechanics/molecular mechanics (QM/MM) on complete enzyme structures and QM cluster models on active site structures only. These two approaches are reviewed here. We give examples where the QM cluster approach worked well and, for instance, enabled the bioengineering of an enzyme to change its functionality. In addition, several examples are given, where QM cluster models were insufficient and full QM/MM structures were needed to establish regio-, chemo-, and stereoselectivities