The phosphonomethyl analogue of 3-phosphoglycerate is a potent competitive inhibitor of phosphoglycerate mutases

Abstract

The phosphonomethyl analogue of 3-phosphoglycerate (2-hydroxy-4-phosphonobutanoate) is a potent competitive inhibitor of cofactor-dependent phosphoglycerate mutase from yeast and of cofactor-independent phosphoglycerate mutase from wheat germ. For the yeast enzyme Ki is 1.3 mM (Km for substrate is 0.71 mM); for the wheatgerm enzyme Ki is 18 mM (Km for substrate is 0.86 mM). This analogue should be a useful tool for n.m.r. spectroscopic studies on the mechanism of action of the two mutases. The arsonomethyl analogue of 3-phosphoglycerate (4-arsono-2-hydroxybutanoate) was a relatively poor inhibitor.

Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase

“…(Sasaki et al, 1971). The affinity constants of the wild-type enzyme for 2,3-bisphosphoglycerate and 3-phosphoglycerate also agree well with those found previously McAleese et al, 1985). The H181A mutation has drastically reduced, but not abolished the activity of phosphoglycerate mutase.…”

The Phosphoglycerate Mutases

“…This cofactor-independent enzyme has been less well characterized than the cofactor-dependent mutases, and relatively little is known of its structure. However, many aspects of its activity have been studied, especially using the enzyme isolated from wheat germ (Britton et al, 1971;Breathnach and Knowles, 1977;McAleese et al, 1985) and from castor oil seeds (Botha and Dennis, 1986). Cofactor-independent phosphoglycerate mutase is active as a monomer which is much larger (Mr about 60,000) than the subunit of the cofactor-dependent enzyme.…”

Do metal ions promote the re-activation of the 2,3-bisphosphoglycerate-independent phosphoglycerate mutases?

“…with 10 mM-3-phosphoglycerate, 3 mM-MgSO4 and 10 j,g of enolase in 0.03 M-Tris/HCl buffer, pH 7.0 (total volume 1.0 ml). A unit of enzyme activity causes an increase in A240 of 0.1 /min under the stated conditions; according to McAleese et al (1985), this corresponds to the conversion of 0.084,mol of 3-phosphoglycerate/min. Phosphoglycerate mutase from rabbit muscle was assayed as described by Grisolia (1962).…”

“…Although this value is comparable with the previous results (Leadlay et al, 1977;Smith & Hass, 1985), the preparations showed various minor bands of lower Mr on SDS/polyacrylamide-gel electrophoresis (Laemmli, 1970) in addition to the major band at an Mr of 60000 + 3000. The presence of these minor bands has been referred to previously (Smith & Hass, 1985;McAleese et al, 1985). Phosphoglycerate mutase from Neurospora crassa Phosphoglycerate mutase, purified from N. crassa, was kindly given by Dr. S. McAleese, Department of Biochemistry, University of Aberdeen, Aberdeen, U.K.…”