The Kinetic Properties of Spinach Leaf Glyoxylic Acid Reductase

Leonard D Kohn,William A Warren

doi:10.1016/s0021-9258(18)62926-6

Abstract

Abstract The kinetic properties of spinach leaf glyoxylic acid reductase have been evaluated. In the presence of pyridine nucleotides, the enzyme catalyzes the reversible reduction of glyoxylate to glycolate and hydroxypyruvate to d(-)-glycerate. The pH optima of the reductive reactions are between 6.0 and 6.5; the pH optima of the oxidative reactions are at 8.9. The enzyme is competitively inhibited by pyruvate, 3-mercaptopyruvate, and 3-fluoropyruvate and noncompetitively inhibited by dihydroxyfumarate. A spectral shift in protein absorbance, from 280 mµ to 270 mµ, is associated with the dihydroxyfumarate inhibition. Ternary rate equations of the enzyme and a degraded form of the enzyme have been calculated and compared. Specific changes in the Michaelis constant for pyridine nucleotides suggest that the degraded form has undergone structural modifications in the pyridine nucleotide binding site. All forms of the enzyme are anion-regulated in the direction of d(-)-glycerate or glycolate formation. Phosphate stimulates hydroxypyruvate reduction at all anion concentrations; chloride, bromide, sulfate, and nitrate stimulate at low concentrations but inhibit at high. The anion effects are pH-dependent and are competitive with hydroxypyruvate or glyoxylate. They are noncompetitive with reduced pyridine nucleotides. Evidence is presented which suggests that a ligand-induced conformational change is important in the functioning of anion effectors.

Highlights

The kinetic properties of spinach leaf glyoxylic acid reductase have been evaluated
The RF 0.19 isozyme was identical with the RF 0.22 enzyme in all respects, i.e. in its substrate specificity, pH optima, salt effects, ternary kinetics, and rate equations
Spinach leaf glyoxylic acid reductase is similar to several bacterial enzymes capable of the catalytic reduction of hydroxypyruvate, i.e. hydroxypyruvate reductase (2), tartronic semialdehyde reductase (3), and oxaloglycolate-reductive decarboxylase (4)

Summary

SUMMARY

The kinetic properties of spinach leaf glyoxylic acid reductase have been evaluated. In the presence of pyridine nucleotides, the enzyme catalyzes the reversible reduction of glyoxylate to glycolate and hydroxypyruvate to D( -)-. The present report characterizes the kinetic properties of this enzyme It shows that the products of hydroxypyruvate and glyoxylate reduction are D( -)-glycerate and glycolate, respectively. The kinetic properties of the enzyme are compared to the kinetic properties of three additional forms of the protein which have been isolated and characterized (1) Two of these forms are considered isozymic in nature since they have the same molecular weight and amino acid composition as the enzyme above. The third is concluded to be a degraded form of the enzyme since it has a lower molecular weight and a different amino acid content (1) These forms have been shown to be readily distinguished by their specific activities, isoelectric points, and stabilities

AND METHODS

RESULTS

Findings

X 1Cr6

DISCUSSION