Purification and Regulatory Properties of the Biosynthetic L-Glycerol 3-Phosphate Dehydrogenase from Escherichia coli

M Kito,L I Pizer

doi:10.1016/s0021-9258(18)93129-7

Abstract

Abstract The l-glycerol-3-phosphate:NAD(P) oxidoreductase (EC 1.1.1.8), referred to as glycerol phosphate dehydrogenase, was purified from extracts of Escherichia coli by a combination of chromatographic procedures. The over-all purification was approximately 1000-fold and the yield was about 10%. The elution profile from the final DEAE-Sephadex column and zone electrophoresis indicated that the enzyme preparation was not homogenous. Kinetic studies showed that the enzyme was both stabilized and inhibited by 0.1 to 1.0 m salts, and that l-glycerol 3-phosphate specifically inhibited dihydroxyacetone phosphate reduction. The curve describing the degree of inhibition as a function of l-glycerol 3-phosphate concentration was sigmoid, with 35 µm glycerol 3-phosphate producing 50% inhibition. Michaelis-Menten type kinetics were found with glycerol 3-phosphate, acting as a substrate, dihydroxyacetone phosphate, and TPNH. The calculated Km values were 210 µm, 170 µm, and 10 µm, respectively. Glycerol 3-phosphate was a competitive inhibitor with respect to dihydroxyacetone phosphate and an uncompetitive inhibitor with respect to TPNH. The addition of TPNH to the enzyme solution quenched the fluorescence of the protein at 340 mµ and enhanced the TPNH fluorescence at 450 mµ. Glycerol 3-phosphate further enhanced the fluorescence of the TPNH-enzyme complex in the 430 to 440 mµ region. The fluorimetric titration of the TPNH-enzyme complex with glycerol 3-phosphate gave a sigmoid curve with the same characteristics as the inhibition curve. The inhibition of the pyridine nucleotide-linked glycerol phosphate dehydrogenase of Escherichia coli, whose most likely function is to provide glycerol 3-phosphate for lipid syntheses, could act in the cell as an effective mechanism for the restriction of the pool size of glycerol phosphate.

Highlights

1.1.1.8). referred to as glycerol phosphate dehydrogenase, of L-glycerol-3-P’ by long chain acyl-CoA [5, 6] or possibly acyl was purified from extracts of Escherichia coli by a combina- carrier protein [6]
The curve and the inhibition of acetyl-CoA carboxylase by long chain acyldescribing the degree of inhibition as a function of L- CoA has been suggested as a mechanism of end product control glycerol 3-phosphate concentration was sigmoid, with [8]
Purity of Enzyme-While the enzyme was purified by the procedure described approximately lOOO-fold it was apparent from the elution profile shown in Fig. 1C that a homogenous preparation was not obtained

Summary

SUMMARY

Referred to as glycerol phosphate dehydrogenase, of L-glycerol-3-P’ by long chain acyl-CoA [5, 6] or possibly acyl was purified from extracts of Escherichia coli by a combina- carrier protein [6]. Kinetic studies showed duce the water-soluble substrates are regulated and, if so, whether that the enzyme was both stabilized and inhibited by 0.1 these compounds are produced in a coordinated way. The properties of the enzyme suggest that was a competitive inhibitor with respect to dihydroxyacetone glycerol-P regulates its own synthesis in the cell by acting on this phosphate and an uncompetitive inhibitor with respect to enzyme and that the L-glycerol-3-P dehydrogenase is an allosteric.

AND METHODS

RESULTS

Third DEAE-Sephadex

DISCUSSION