A general kinetic approach to investigation of active-site availability in macromolecular catalysts

A potentially general kinetic method for the investigation of active-site availability in preparations of macromolecular catalysts was developed. Three kinetic models were considered: (a) the conventional two-step model of enzyme catalysis, where the preparation contains only active catalyst (E_a) and inert (i.e. non-binding, non-catalytic) material (E_i); (b) an extension of the conventional model (a) involving only E_a and E_i, but with non-productive binding to E_a (in addition to productive binding); (c) a model in which the preparation contains also binding but non-catalytic material (E_b), predicted to be present in polyclonal catalytic antibody preparations. The method involves comparing the parameters V_max and K_m obtained under catalytic conditions where substrate concentrations greatly exceed catalyst concentration with those (k, the limiting value of the first-order rate constant, k_obs, at saturating concentrations of catalyst; and K) for single-turnover kinetics, in which the reverse situation obtains. The active-site contents of systems that adhere to model (a) or extensions that also lack E_b, such as the non-productive binding model (b), may be calculated using [E_a]_T = V_max/k. This was validated by showing that, for α-chymotrypsin, identical values of [E_a]_T were obtained by the kinetic method using Suc-Ala-Ala-Pro-Phe-4-nitroanilide as substrate and the well-known ‘all-or-none’ spectroscopic assay using N-trans-cinnamoylimidazole as titrant. For systems that contain E_b, such as polyclonal catalytic antibody preparations, V_max/k is more complex, but provides an upper limit to [E_a]_T. Use of the kinetic method to investigate PCA 271-22, a polyclonal catalytic antibody preparation obtained from the antiserum of sheep 271 in week 22 of the immunization protocol, established that [E_a]_T is less than approx. 8% of [IgG], and probably less than approx. 1% of [IgG].