Observation of a kinetic slow transition in monomeric glucokinase.
Rat liver glucokinase (EC 2.7.1.2) is a monomeric enzyme with positive cooperativity for glucose phosphorylation for which several kinetic mechanisms have been proposed. We have observed a slow kinetic transition when the enzyme is assayed in the presence of 30% glycerol. When the enzyme had been preincubated or stored in 50 mM glucose, the initially rapid activity decayed, via a first-order process, to a new steady-state velocity. The glucose-induced process is reversible since if the enzyme is preincubated without glucose, an initially low activity accelerates over minutes to the same steady-state velocity. This final velocity is independent of the preincubation conditions and is determined solely by the glucose and ATP concentrations in the assay. Possible artifacts which might cause nonlinear progress curves have been ruled out. The transition has a half-time of 2-10 min depending on glucose and ATP concentrations and temperature. In the steady-state kinetics, positive cooperativity occurs with glucose with a Hill coefficient (nH) = 1.3 at high ATP concentrations, approaching unity as the ATP concentration decreases. This pattern is similar to that seen in the linear velocities in the absence of glycerol. Similarly, negative cooperativity with MgATP is seen in the steady-state velocities at low glucose concentrations with the Hill coefficient approaching 1 as the glucose concentrations approach saturation. The initial velocity for enzyme preincubated in high glucose concentration was either Michaelis-Menten as a function of glucose at high MgATP concentration or heterogeneous (nH less than 1, negatively cooperative) at low MgATP concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Neet
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Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Keenan
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RP
Tippett
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D000117:Acetylglucosamine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D005941:Glucokinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D013379:Substrate Specificity
D007527:Isoenzymes; D000255:Adenosine Triphosphate; D005941:Glucokinase; D005990:Glycerol; D000117:Acetylglucosamine