Kinetics and thermodynamics of activation by pretreatment with guanosine 5′-[βγ-imido]triphosphate of smooth-muscle adenylate cyclase

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Abstract
Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5′-[βγ-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2°C. Activation (C→C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37°C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37°C. Over the range 6–24°C, activation by pretreatment with p[NH]ppG followed simple Michaelis–Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased Km for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (37°C) was moderately endergonic (ΔH0 ∼8kJ·mol−1) and accompanied by an increase in entropy (ΔS0 ∼146J·mol−1·K−1). The β-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P>0.05) effect on the Km at every temperature. This result suggested that formation of the receptor–hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the β-adrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.

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