Preferential binding of competitive inhibitors to the monomeric form of α-chymotrypsin

Abstract

in phosphate (/ = 0.28, pH 7.8) have been interpreted quantitatively in terms of a monomer-dimer acceptor system, the association constant for the interaction of phenylpropiolate with the two binding sites on the dimeric form of a-chymotrypsin being approximately four-fifths of the corresponding value for the single site on monomeric enzyme. These results, pertaining to the inhibitor-binding properties of a-chymotrypsin in solution correlate well with published X-ray crystallographic data, which indicate that the approach of substrate analogs to the active sites of dimeric -chymotrypsin is subject to steric hindrance.

Foreword to ‘Quantitative and analytical relations in biochemistry’—a special issue in honour of Donald J. Winzor’s 80th birthday

“…Collaborative studies with Laurie Nichol continued, with demonstrations of ways in which to unravel and quantify the interactions responsible for various types of self-association ) and binding behaviour (Baghurst et al 1971;Nichol et al 1972Nichol et al , 1973Winzor et al 1977;Tellam et al 1978;Ward and Winzor 1982;Cann and Winzor 1987;Gow et al 1990;Wilson et al 1997;Winzor et al 1998). The effect of ligand-induced self-association on the size of acceptor species (Nichol and Winzor 1976) was used to explore Don's undergraduate lecturing and postgraduate supervision were notable for a number of things.…”

The development of chromatography for the characterization of protein interactions: a personal perspective

“…The consequent displacement of the acceptor equilibrium position by preferential ligand binding may lead to a relatively minor change in the proportions of monomer and oligomeric states, and hence to only a small change in the elution volume of the protein constituent (V A ). Difference gel chromatography was therefore developed [19,20] to facilitate with phenylpropiolate to a Sephadex G-75 column equilibrated with ligand-free buffer (pH 7.8, I 0.28) -an experiment designed to detect preferential ligand interaction with monomeric enzyme by difference gel chromatography. Data taken from [20].…”

“…Difference gel chromatography was therefore developed [19,20] to facilitate with phenylpropiolate to a Sephadex G-75 column equilibrated with ligand-free buffer (pH 7.8, I 0.28) -an experiment designed to detect preferential ligand interaction with monomeric enzyme by difference gel chromatography. Data taken from [20].…”

“…The principle of difference gel chromatography is illustrated by a study designed to demonstrate the preferential binding of phenylpropiolate (a competitive inhibitor) to monomeric α-chymotrypsin [20]. Figure 2(b) shows the advancing elution profile obtained by applying α-chymotrypsin solution supplemented with 20 mM phenylpropiolate to a Sephadex G-75 column pre-equilibrated with ligand-free buffer.…”

“…For phenylpropiolate the degree of preference for binding to monomeric α-chymotrypsin is relatively small in that K AS for dimeric enzyme is 80 % of that for monomer [20].…”