The Kinetic Mechanism of Glycerokinase

The steady state kinetics of glycerokinase from Candida mycoderma has been studied at pH 7.0 by initial velocity and product inhibition techniques and by dead-end inhibition by the inert coordination complex, chromium ATP. The mechanism appears to be Ordered Bi Bi with glycerol adding before the magnesium nucleotide and l-glycerol 3-phosphate being released last. With different nucleotides as substrates the dissociation constant for glycerol is constant, but the Kms of the nucleotides vary from 9 µm for MgATP to 2400 µm for MgXTP. In addition to glycerol (Km, 15 µm), dihydroxyacetone (Km, 5 mm) and l-glyceraldehyde (Km, 3.7 mm) are phosphorylated. d-Glyceraldehyde (Km, 0.5 mm) is not phosphorylated but incluces hydrolysis of MgATP. Glycerol is competitive versus l-glycerol 3-phosphate and noncompetitive versus MgADP. CrATP is competitive versus MgATP and is uncompetitive versus glycerol or MgADP. The dissociation constant of CrATP from the enzymeglycerol complex is 0.5 to 0.7 µm. In an ordered mechanism the sum of the vertical coordinates of the crossover points in initial velocity patterns for forward and reverse reactions, divided by the sum of reciprocal velocities in both directions, is a dimensionless number varying from 0 for a mechanism where second product release is solely rate limiting, to 1 for a rapid equilibrium ordered mechanism where catalysis and/or first product release is solely rate limiting. Where the two maximum velocities are not equal, this number indicates the rate-limiting step in the slower direction only. For glycerokinase this analysis and comparison of inhibition constants for CrATP versus MgATP and MgADP show that in the reverse direction, glycerol release is 2 to 5 times faster than catalysis and/or release of MgATP.