Enzymatic activities of human cytomegalovirus maturational protease assemblin and its precursor (pPR, pUL80a) are comparable: [corrected] maximal activity of pPR requires self-interaction through its scaffolding domain.
Herpesviruses encode an essential, maturational serine protease whose catalytic domain, assemblin (28 kDa), is released by self-cleavage from a 74-kDa precursor (pPR, pUL80a). Although there is considerable information about the structure and enzymatic characteristics of assemblin, a potential pharmacologic target, comparatively little is known about these features of the precursor. To begin studying pPR, we introduced five point mutations that stabilize it against self-cleavage at its internal (I), cryptic (C), release (R), and maturational (M) sites and at a newly discovered "tail" (T) site. The resulting mutants, called ICRM-pPR and ICRMT-pPR, were expressed in bacteria, denatured in urea, purified by immobilized metal affinity chromatography, and renatured by a two-step dialysis procedure and by a new method of sedimentation into glycerol gradients. The enzymatic activities of the pPR mutants were indistinguishable from that of IC-assemblin prepared in parallel for comparison, as determined by using a fluorogenic peptide cleavage assay, and approximated rates previously reported for purified assemblin. The percentage of active enzyme in the preparations was also comparable, as determined by using a covalent-binding suicide substrate. An unexpected finding was that, in the absence of the kosmotrope Na2SO4, optimal activity of pPR requires interaction through its scaffolding domain. We conclude that although the enzymatic activities of assemblin and its precursor are comparable, there may be differences in how their catalytic sites become fully activated.
Brignole
Edward J
EJ
Virology Laboratories, The Department of Pharmacology & Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. wgibson@jhmi.edu
Gibson
Wade
W
D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003587:Cytomegalovirus / Q000201:enzymology*; D003956:Dialysis; D004792:Enzyme Precursors / Q000378:metabolism*; D005470:Fluorometry; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D011489:Protein Denaturation; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*
D004792:Enzyme Precursors; D010455:Peptides; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C071910:assemblin