Deacylation and reacylation for a series of acyl cysteine proteases, including acyl groups derived from novel chromophoric substrates

In order to investigate structure-reactivity relationships within a series of acyl cysteine proteases [Doran, J. D., & Carey, P. R. (1996) Biochemistry 35, 12495-12502], deacylation kinetics have been measured for a number of acyl intermediates involving members of the papain superfamily. Derivatives of the ''simple'' chromophoric ligand (5-methylthienyl)acrylate (5MTA) and those based on two chromophorically labeled derivatives of peptidyl substrates, viz., 2-[(N-acetyl-L-phenylalanyl)amino]-3-(5-methylthienyl)acrylate (Phe5MTA) and 2-[(N-acetyl-L-alanyl)amino]-3-(5-methylthieny)acrylate (Ala5MTA), were used to create acyl enzyme adducts with papain, cathepsin B, and cathepsin L. The chromophoric specific substrates were designed to utilize hydrogen-bonding and hydrophobic interactions which are known to be important in promoting catalysis by papain. For cathepsins B and L, removing one of the hydrogen-bonding donors making up the putative oxyanion hole retards deacylation by 3-25-fold, demonstrating that the oxyanion hole has a modest effect on catalysis for these substrates. With the above substrates and the wild-type and oxyanion hole mutants, the values of the deacylation rate constants stretch over a 214-fold range, from 0.07 to 15 x 10(-3)s(-1). The pK(a) for deacylation of [(5-methylthienyl)acryloyl]papain is 4.9, close to that reported for similar papain intermediates, while that for Ala5MTA papain is at 3.5, which in the latter likely represents the effect of the P-1-S-1 and P-2-S-2 interactions on the environment of histidine-159. For the Phe5MTA-papain the extent of deacylation was found to depend on the pH and the starting acyl enzyme concentration A simple model has been derived which accounts quantitatively for this behavior, using the assumptions that the protonated form of the acyl product reacylates the enzyme and that in the pH range 5.0-7.5 the ionization of active-site groups has no effect on reacylation. The validity of the first assumption was demonstrated by following the deacylation of Phe5MTA-papain in the presence of the potent inhibitor E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane], whereupon complete deacylation occurred at all pHs with a pK(a) identical to that for Ala5MTA-papain, viz., 3.5.