The pH-dependence of rates of individual steps in ficin catalysis.

Abstract

The kinetics of the ficin‐catalysed hydrolysis of benzoylglycine p‐nitrophenyl ester have been studied at different pH values with widely varying initial concentrations of enzyme and substrate. The time‐courses of proton release and p‐nitrophenol release were followed. The failure to observe a lag period for p‐nitrophenol release employing a stopped‐flow spectrophotometer leads to a minimum value for the rate constant for the formation of the E · S complex of 5 × 105 M−1 sec−1. Evidence is presented for the formation of E · S being a pseudo‐equilibrium process. The results are consistent with a three‐step mechanism involving an acyl‐enzyme intermediate. Time‐courses of reactions with [E0] ∼ [S0] were independent of wavelength in the range 291 to 400 nm so that it is unlikely that an oxazolinone is formed as an intermediate. The pH‐dependencies have been determined for the dissociation constant of the E · S complex (Ks) as well as for the individual rate constants associated with the catalytic steps (k+2 and k+3). pH profiles of k+2/Ks implicate ionising groups of pKa values 4.3 and 8.58 in the free enzyme (25 °C, I= 0.1). The pH dependency of k+2 (measured directly at pH values below neutrality) implicates an ionising group in the catalytic step with pKa∼ 5.8. The change in the value of Ks with pH is in accord with this being identical to the group with pK 4.3 in the free enzyme. The value of k+3 does not vary significantly between pH 3.9 and 6.6, from which it is deduced that the group of pKa∼ 5.8 in the E · S complex undergoes a downward shift in pKa by at least 2 units on formation of the acyl‐enzyme intermediate. The release of protons concomitant with acyl‐enzyme formation (pH 5 and [E0] ∼ [S0]) is consistent with such a pKa shift. At neutral pH values the k+3 step is rate limiting in catalysis whereas at pH 3.9 k+2 becomes rate limiting. This change in rate‐limiting step with pH illustrates the danger in the assumption that kcatversus pH profiles for thiol protease action on substrates with good leaving groups are equivalent to k+3versus pH profiles.