Kinetic analysis and chemical modification of vitamin B6 phosphatase from human erythrocytes.
The specificity and active site properties of vitamin B6 phosphatase purified from human erythrocytes were studied by kinetic analyses with vitamin B6 compounds and derivatives and chemical modification with group-specific reagents. The kinetic constants for pyridoxal phosphate (PLP), 4-pyridoxic acid phosphate, pyridoxine phosphate, and pyridoxamine phosphate were determined from pH 5 to 9. The values of Vmax/Km and pKm were highest for PLP and 4-pyridoxic acid phosphate and lowest for pyridoxamine phosphate. Vmax/Km and pKm for the four substrates were maximum between pH 6 and 8. Ionizable groups with pKa values about 6 and 8 affected substrate binding to the enzyme. Vmax values for all the substrates gradually decreased with increasing pH. The enzyme also catalyzed the dephosphorylation of 4'-secondary amine derivatives of vitamin B6-phosphate. The phosphatase had greatest catalytic efficiency with substrates that contained a negatively charged group on the 4'-position of the pyridine ring. It is concluded that there are one or two positively charged groups at the active site of the enzyme that interact with the substrate's phosphate ester and 4'-substituent. The phosphatase was inactivated by phenylglyoxal, and PLP protected the enzyme against this inactivation. Phenylglyoxal did not modify Lys or Cys residues or an alpha-amino group since the enzyme's NH2 terminus is blocked, and it did not affect the quaternary structure of the phosphatase. The enzyme was inactivated by the incorporation of 1 mol of phenylglyoxal/subunit. Diethylpyrocarbonate inactivated the enzyme by reacting with a group with a pKa of 6.7, and pyridoxine phosphate protected the enzyme against this inactivation. These data suggest that Arg and His residues are at or near the active site and may play roles in substrate binding and/or catalysis.
Gao
G J
GJ
Department of Biochemistry, University of Louisville School of Medicine, Kentucky 40292.
Fonda
M L
ML
D004047:Diethyl Pyrocarbonate; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D010658:Phenylglyoxal; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity; D013774:Tetranitromethane
C027138:pyridoxine phosphate phosphatase; D010744:Phosphoric Monoester Hydrolases; D013774:Tetranitromethane; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal