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 (Ea) and inert (i.e. non-binding, non-catalytic) material (Ei); (b) an extension of the conventional model (a) involving only Ea and Ei, but with non-productive binding to Ea (in addition to productive binding); (c) a model in which the preparation contains also binding but non-catalytic material (Eb), predicted to be present in polyclonal catalytic antibody preparations. The method involves comparing the parameters Vmax and Km obtained under catalytic conditions where substrate concentrations greatly exceed catalyst concentration with those (k, the limiting value of the first-order rate constant, kobs, 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 Eb, such as the non-productive binding model (b), may be calculated using [Ea]T = Vmax/k. This was validated by showing that, for α-chymotrypsin, identical values of [Ea]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 Eb, such as polyclonal catalytic antibody preparations, Vmax/k is more complex, but provides an upper limit to [Ea]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 [Ea]T is less than approx. 8% of [IgG], and probably less than approx. 1% of [IgG].
A general kinetic approach to investigation of active-site availability in macromolecular catalysts
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Marina RESMINI, Sheraz GUL, Steve CARTER, Sanjiv SONKARIA, Christopher M. TOPHAM, Gerrard GALLACHER, Keith BROCKLEHURST; A general kinetic approach to investigation of active-site availability in macromolecular catalysts. Biochem J 15 February 2000; 346 (1): 117–125. doi: https://doi.org/10.1042/bj3460117
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