Abstract
Rat liver adenosine kinase can catalyze an exchange reaction between adenosine and AMP in the absence of ATP (Bontemps, F., Mimouni, M., and Van den Berghe, G. (1993) Biochem. J. 290, 679-684), suggesting a classical ping-pong mechanism. Contrary to expectations, formation of a phosphorylenzyme intermediate could not be demonstrated by incubating the enzyme with [gamma-32P] ATP. Although initial velocity measurements in function of the concentration of adenosine or Mg.ATP, at various fixed concentrations of Mg.ATP or adenosine, generated parallel line patterns, inhibition studies revealed that competitive inhibition was only observed between ADP and ATP. This indicates an Ordered Bi Bi mechanism in which ATP binds first to the enzyme, and ADP is released last. The adenosine-AMP exchange reaction was found to be potently stimulated by ADP, and the basal exchange reaction, i.e. measured in the absence of added ADP, could be accounted for by a slight (0.001%) contamination by ADP of analytical grade AMP. The ADP requirement of the adenosine-AMP exchange reaction explains its occurrence in an Ordered Bi Bi mechanism. Stimulation of the exchange reaction between AMP and adenosine by increasing concentrations of ADP/ATP, and stimulation followed by inhibition of the exchange reaction between ADP and ATP by increasing concentrations of AMP/adenosine, corroborated the proposed mechanism.
Highlights
From the Laboratory of Physiological Chemistry, International Instituteof Cellular a n d Molecular Pathology and the University of Louvain Medical School, B-1200 Brussels,Belgium
Phosphorylation of Adenosine Kinase-As explained in the Introduction, the observation that rat liver adenosine kinase can catalyze an exchange reaction between adenosineAaMnPd in the absence of ATP suggested a classical ping-pong mechanism
Adenosine, no radioactivity was found in the protein, either collected on Whatman P-81 papoerrisolated by Sephadex G-25
Summary
(Received for publication, February 3, 1994, and in revised form, March 31, 1994). From the Laboratory of Physiological Chemistry, International Instituteof Cellular a n d Molecular Pathology and the University of Louvain Medical School, B-1200 Brussels,Belgium. Rat liver adenosine kinase can catalyze an exchange AMP the last product to be released. EC 2.7.1.20) is an ubiquitous enzymethat catalyzes the phos- (Mannheim,Germany).Dye reagent concentrate,bovine y-globulin,and phorylation of adenosine andof several related nucleosides andthe SDS-polyacrylamide gel electrophoresis M, standards werefrom analogs intothe corresponding monophosphates, using preferentially ATP or GTaPs the phosphoryl donor (Anderson 973). EC 2.7.1.20) is an ubiquitous enzymethat catalyzes the phos- (Mannheim,Germany).Dye reagent concentrate,bovine y-globulin,and phorylation of adenosine andof several related nucleosides andthe SDS-polyacrylamide gel electrophoresis M, standards werefrom analogs intothe corresponding monophosphates, using preferentially ATP or GTaPs the phosphoryl donor (Anderson1, 973) It is a key enzyme in the control of the concentration of adenosine, an important physiological regulatorin several tissues (Arch and Newsholme, 1978; Fox a n d Kelley, 1978). Each experiment was performed at least 3-fold with variations of less than 10%.Kinetic constants were graphically derived from replots of slopes and intercepts
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