Glutamate 325 is a general acid-base catalyst in the reaction catalyzed by fructose-2,6-bisphosphatase.
A bifunctional enzyme, fructose-6-phosphate, 2-kinase:fructose-2, 6-bisphosphatase, catalyzes synthesis and hydrolysis of fructose 2, 6-bisphosphate. The phosphatase reaction occurs in two steps: the formation of a phosphoenzyme intermediate and release of beta-D-fructose 6-phosphate, followed by hydrolysis of the phosphoenzyme. The objective of this study was to determine whether E325 in the Fru 2,6-Pase active site is an acid-base catalyst. The pH-rate profile for k(cat) for the wild-type enzyme exhibits pK values of 5.6 and 9.1. The pH dependence of k(cat) for the E325A mutant enzyme gives an increase in the acidic pK from 5.6 to 6.1. Formate, acetate, propionate, and azide accelerate the rate of hydrolysis of the E325A mutant enzyme, but not of the wild-type enzyme. Azide and formate, the smallest of the weak acids tested, are the most potent activators. The k(cat) vs pH profile of the E325A mutant enzyme in the presence of formate is similar to that of the wild-type enzyme. Taken together, these data are consistent with E325 serving an acid-base role in the phosphatase reaction. The exogenous low MW weak acids act as a replacement general base in the hydrolysis of the phosphoenzyme intermediate, rescuing some of the activity lost upon eliminating the glutamate side chain.
Sakurai
M
M
Research and Development, Dallas Veterans Affairs Medical Center and Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Texas 75216, USA.
Cook
P F
PF
Haseman
C A
CA
Uyeda
K
K
D000409:Alanine / Q000235:genetics; D000818:Animals; D002264:Carboxylic Acids / Q000737:chemistry; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D005561:Formates / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics; D051381:Rats; D019810:Sodium Azide / Q000737:chemistry; D013737:Testis
D002264:Carboxylic Acids; D005561:Formates; C030544:formic acid; D018698:Glutamic Acid; D019810:Sodium Azide; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D000409:Alanine