Static and Kinetic Studies on the Binding of Streptomyces Trehalase Inhibitor SGI with Rhizopus Glucoamylase. Comparison with Glucose and Gluconolactone1

Abstract

The binding mechanism of Streptomyces trehalase inhibitor SGI and Rhizopus glucoamylase was investigated by the following approaches; inhibitory kinetic study in the steady-state, fluorometric titration, UV difference spectrophotometry, and stopped-flow kinetic study with fluorescence monitoring. The inhibition by SGI of the hydrolysis of p-nitrophenyl α-D-glucoside was found to be of the mixed type with an inhibitor constant K₁ = 16.4 μM (pH 4.5, 25°C). From fluorometric titration of the enzyme with SGI, the dissociation constant of the enzyme-SGI complex, K_d, was estimated to be 24.μM (pH 4.5, 10°C). The thermodynamic quantities were obtained from the temperature dependence of K_d, and a large positive entropy change, 74 J·mol⁻¹(18 e.u.), and a small negative enthalpy change, −4.2 kJ·mol⁻¹ (−1.0 kcal·mol⁻¹), were noted. The pH dependence of K_d suggested that the binding reaction involves one ionizable group of pK_e=5.8 (both at 5°C and at 25°C). The fluorometric titration of the enzyme with gluconic acid-1,5-lactone in the presence of SGI suggested the binding of SGI to the nonreducing-end terminal subsite (Subsite 1) of the enzyme (Hiromi et al. (1973) Biochim. Biophys. Acta302, 362). The UV absorption difference spectrum of the enzyme caused by the binding of SGI was similar to that caused by gluconic acid 1,5-lactone, except that the trough near 300 nm is not as clear in the case of SGI as in the case of gluconic acid-1,5-lactone. Fluorescence stopped-flow kinetics of the binding was consistent with a two-step mechanism in which a fast bimolecular association is followed by a slow unimolecular process. The dissociation constant of the fast bimolecular step was estimated to be 4.9 mm and the forward and the backward rate constants of the unimolecular process were determined to be 220 s⁻¹ and 1.1 s⁻¹, respectively. It was found that the decrease in the enzyme fluorescence due to the binding of SGI occurs solely in the unimolecular process.