Catalytic activity of the serine proteases alpha-chymotrypsin and alpha-lytic protease tagged at the active site with a (terpyridine)platinum(II) chromophore.
Incubation of alpha-chymotrypsin and alpha-lytic protease with chloro(2,2':6',2''-terpyridine)platinum(II), [Pt(trpy)Cl]+, results in attachment of Pt(trpy)2+ tags at both His 57 and His 40 in the former and His 57 in the latter. The [Pt(trpy)His]2+ chromophores are readily detected and quantitated owing to their characteristic, strong UV absorption. Although the tagging of His 57 modifies the catalytic triad (Ser 195, His 57, and Asp 102) and disrupts the charge relay, the platinated enzymes retain significant esterase and amidase activity for both specific and nonspecific substrates. Unlike suicide inhibitors, which inactivate the enzymes by filling the active site and imitating the tetrahedral intermediate, [Pt(trpy)Cl]+ reacts with a particular amino acid and permits binding of substrates. The kinetic constants for the following are reported: two esters and two amides with alpha-chymotrypsin and an amide with alpha-lytic protease. The kcat values are between 1 and 25% of, and the Km values are a little higher than, the corresponding values for the native enzymes. The catalytic activity is not due to the native enzymes, trypsin, or some zinc-containing protease. Activities of the native and of the platinated alpha-chymotrypsin depend similarly on pH although the pKa of His 57 is raised to 9.7 upon platination. The platinated enzymes undergo autodigestion slower than do the native enzymes. Because the Pt(trpy)2+ tags are noninvasive, stable, and yet easily removable by thiourea, [Pt(trpy)Cl]+ may be used to retard autodigestion of stored proteolytic enzymes.
Brothers
H M
HM
Department of Chemistry, Iowa State University, Ames 50011.
Kostić
N M
NM
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D004950:Esterases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009944:Organoplatinum Compounds; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity
D009944:Organoplatinum Compounds; C021975:chloroterpyridineplatinum(II); D004950:Esterases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C021826:myxobacter alpha-lytic proteinase; D000581:Amidohydrolases; C019665:amidase