Substrate Modulation of Enzyme Activity in the Herpesvirus Protease Family

Abstract

The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 A2 of surface are buried in the newly formed S4 pocket when tyrosine binds at this site. The peptide inhibitor also induces a rearrangement of residues that stabilizes the oxyanion hole and the dimer interface. Concomitant with the structural changes, an increase in catalytic efficiency of the enzyme results upon extended substrate binding. A nearly 20-fold increase in kcat/KM results upon extending the peptide substrate from a tetrapeptide to a hexapeptide exclusively due to a KM effect. This suggests that the mechanism by which herpesvirus proteases achieve their high specificity is by using extended substrates to modulate both the structure and activity of the enzyme.

(±)-Japonones A and B, two pairs of new enantiomers with anti-KSHV activities from Hypericum japonicum

“…The crystal structures of the docking targets including KSHV protease, KSHV LANA, PKC, P38, JNK, and ERK were obtained from the Protein Data Bank (http://www.rcsb.org)232425262728. Prior to the docking simulations, the structures of the compounds were visualized using ChemOffice 12.0 (CambridgeSoft), and the minimum energy conformation of each compound was determined using the standard Tripos molecular mechanics force field in the SYBYL-X 2.0 molecular modelling package.…”

Broad-Spectrum Allosteric Inhibition of Herpesvirus Proteases

“…6,12,13,16−26 Each monomer has an independent active site. 1 In the monomeric state, the enzyme is inactive and partially disordered.…”

A Screening Strategy for Trapping the Inactive Conformer of a Dimeric Enzyme with a Small Molecule Inhibitor

“…The peptide sequence is Ac—Pro—Val—Tyr—tBug—Gln—Ala—ACC with an observed K M of 8.5 ± 0.8 µM for KSHV Pr. Substrate is synthesized using standard FMOC chemistry and purified as reported previously(7,12). Substrate stocks of 10 mM in 100% DMSO are stored at −20 °C.…”

“…Efforts by pharmaceutical companies to target the active-site of the essential dimeric serine protease of human herpesviruses (HHV) have yet to yield a drug-like candidate(1–6). Given the evidence supporting a conformational linkage between protease dimerization and activation, we have focused our efforts on identifying molecules that target the dimer interface(7–12). In the case of KSHV protease (KSHV Pr) the dimer interface covers approximately 2500 Å 2 , and includes the α-helix 5 of each monomer as the major constituent (Figure 1)(13).…”

“…Screening was performed in a 96—well plate format using a fluorogenic activity assay. The substrate used is an optimized hexa—peptide attached to 7—amino—4—carbamoylmethyl coumarin (ACC), where cleavage at the scissile bond releases the ACC group resulting in increased fluorescence(7,12). We have successfully used this approach to identify the small molecule inhibitor DD2 (Figure 3), which binds a novel allosteric pocket at the dimer interface of KSHV Pr, and traps it in an inactive monomeric state(16).…”