Steady‐State Kinetic Studies on Adenylate Cyclase from Brevibacterium liquefaciens

Abstract

Kinetic data of basal adenylate cyclase activity were found to be in agreement with a negatively cooperative model for this enzyme. The model assumes a dimeric structure for the enzyme with two binding sites per dimer. The binding of the first substrate molecule increases the dissociation constant for substrate molecules increases the dissociation constant for substrate molecules at the second site. However, the doubly occupied enzyme has a greater catalytic efficiency per site than the singly occupied species. The effect of various nucleoside 5'-triphosphates on the basal enzymatic activity was investigated. At low concentrations of substrate and analogue relative to K, and K,, compounds closely related to ATP, like 3'-deoxy-adenosine 5'-triphosphate, 2-amino-adenosine 5'-triphosphate and 3'-deoxy-3'-amino-adenosine 5'-triphosphate activate adenylate cyclase. An increase in the concentration of either substrate or analogue reverses the activation effect. GTP, UTP, CTP and 2-aminopurineriboside 5'-triphosphate are purely competitive inhibitors of basal adenylate cyclase activity at low substrate concentrations. This behaviour can be explained by the assumption that coexistence of substrate and inhibitor fails to induce the increase in catalytic efficiency. Kinetic analysis of the activation of adenylate cyclase by pyruvate suggested that the primary effect of the activator is to abolish the negative cooperativity between subunits, at the same time inducing a better fit between the enzyme and its substrate. A tentative model, which is in reasonable agreement with the kinetic data is presented. Mixed inhibition of pyruvate-stimulated adenylate cyclase was observed with GTP. Kinetic analysis revealed that this was the result of normal competitive inhibition of ATP binding, on which an increase of the apparent affinity of the enzyme for pyruvate was superimposed.The adenylate cyclase of Brevihacterium liquefuciens is the first species of its kind to have been purified to crystalline homogeneity [l]. A dimer of molecular weight 92400, it catalyzes the reversible conversion of ATP to cAMP and inorganic pyrophosphate [2,3]. The activity extends to dATP and AdoP [NH]PP, but the enzyme is not active on the other naturally occurring nucleoside triphosphates. A characteristic feature of the enzyme is its stimulation in vitro, as well as in vivo, by 2-0x0-monocarboxylic acids, especially by pyruvate [1,4-81. The present report deals with the kinetic details of pyruvate activation of adenylate cyclase from Brevibacterium liquefuciens. We furthermore investigated the response of pyruvate-stimulated as well as basal adenylate cyclase activities to purine and pyrimidine nucleoside triphosphates as effectors.

Biological and Chemical Bases for the Reclassification of Brevibacterium vitarumen (Bechdel et al.) Breed (Approved Lists, 1980) as Corynebacterium vitarumen (Bechdel et al.) comb. nov. and Brevibacterium liquefaciens Okabayashi and Masuo (Approved Lists, 1980) as Corynebacterium liquefaciens (Okabayashi and Masuo) comb. nov.

“…Although these authors found that pyruvate was required for activity of the crystalline enzyme, they left the mode of interaction between the enzyme and its substrate open to question (49). In a study of the steady-state kinetics of the adenyl cyclase of B. Ziquefaciens Ozer and Scheit suggested that activation by pyruvate involves abolishing "negative cooperativity" between the subunits of the cyclase, with concomitant induction of a better fit of the enzyme for its substrate (38 …”