PH-DEPENDENT EFFECTS OF CR(NH3)2ATP ON KINETICS OF YEAST HEXOKINASE PII - RELATIONSHIP TO SLOW TRANSITION MECHANISM

  • 1 January 1976
    • journal article
    • research article
    • Vol. 251  (23) , 7521-7525
Abstract
The effect of Cr(NH3)2ATP [chromium diamine adenosine triphosphate], a virtually inert, inner sphere metal .cntdot. ligand complex, on the kinetics of purified yeast hexokinase PII was studied at pH 6.5 and pH 7.5. At pH 6.5, where the normal assays exhibit a slow burst-type transient, low concentrations of Cr(NH3)2ATP were found to activate both .PHI.i, the initial velocity, and .PHI.II, the steady state velocity. At higher concentrations, Cr(NH3)2ATP was found to be a competitive inhibitor vs. MgATP for both .PHI.i and .PHI.II. The apparent Ki values for both velocities were the same. The inhibition by Cr(NH3)2ATP at pH 6.5 was a slow process with half-times similar to those found for the normal burst-type transient at this pH value. At pH 7.5, where normal assays exhibit linear process curves, Cr(NH3)2ATP behaved similarly to that observed before at pH 7, i.e., it was a competitive inhibitor vs. MgATP and it caused a slowing of the reaction rate over the first several minutes. The apparent Ki for the initial velocity was 8-fold higher than the apparent Ki for the steady state velocity, suggesting tighter binding of Cr(NH3)2ATP with time. Preincubation experiments indicated that the normal pH 6.5 burst-type transient could be eliminated by appropriate preincubation with Cr(NH3)2ATP and a sugar. In agreement with Danenberg and Cleland, similar preincubations have been shown to produce linear assays at pH 7.5 in the presence of Cr(NH3)2ATP. Similar results were seen with MgITP as the nucleotide substrate, where a burst-type transient is not seen at either pH value under normal assay conditions. At pH 7.5, a slow decrease in the reaction rate is seen over the first several min in the presence of Cr(NH3)2ATP. The apparent Ki for .PHI.i was 7-fold higher than the apparent Ki value for .PHI.II, again suggesting a tighter binding of Cr(NH3)2ATP with time. A similar observation was made at pH 6.5, but the Ki values for .PHI.i and .PHI.II were the same, suggesting no tightening of the binding of Cr(NH3)2ATP with time at this pH value. These results suggested that both slow processes reflect the same basic molecular change, but the consequences are different at the two pH values, presumably because of the difference in the charge of the enzyme. The Cr(NH3)2ATP kinetics at pH 6.5 were interpreted in terms of a modification of the slow transition mechanism for hexokinase. It is postulated that glucose and Cr(NH3)2ATP induce the same slow conformational change at pH 6.5 as that induced by glucose and MgATP, which gives rise to the normal burst-type transient. This suggests that Cr(NH3)2ATP may be a useful tool for physical studies to determine the cause of the slow transition of yeast hexokinase. Activation by low concentrations of Cr(NH3)2ATP was interpreted as binding of the nucleotide to an activator site on the enzyme, causing a shift in the distribution of enzyme towards the more active form.

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