title: Stereospecificity of the metal.ATP complex in flavokinase from rat small intestine.
abstract: Transfer of the gamma-phosphoryl group of ATP to riboflavin is catalyzed by flavokinase, which prefers Zn(II), and is essential in the biosynthesis of the flavocoenzyme, FMN. To study the mechanism and steric disposition of ATP binding, adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) were tested in comparison to ATP with representative divalent metal ions. Activities with 0.1 mM Zn(II) for 1 mM ATP thio analogues compared to ATP with flavokinase were 60% for the S-isomer of ATP beta S, 312% for the R-isomer of ATP beta S, and 14% for ATP gamma S. With Mg(II), flavokinase showed stereospecificity for the R-isomer of ATP beta S with a Vmax ratio, S/R = 0.125. With Cd(II), the enzyme showed preference for the S-isomer of ATP beta S with a Vmax ratio, S/R = 1.261. The Vmax ratio progressively increases from Mg(II) to Cd(II) in the order of affinity for sulfur. The ratios, (Vmax/Km)Mg/(Vmax/Km)Cd, for the diastereomers of ATP beta S were expectedly greater than 1 for one diastereomer (R = 6.597) and less than 1 for the other (S = 0.292). Activities with ATP gamma S were much lower than with ATP or ATP beta S. With Mg(II), the gamma analogue was a good substrate; however, with Cd(II), it was hardly active. Altogether these results indicate that flavokinase uses the lambda, beta, gamma-bidentate Zn.ATP as a substrate.
authors.affiliation: Department of Biochemistry, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: H
authors.initials: H
authors.lastname: Nakano
authors.forename: D B
authors.initials: DB
authors.lastname: McCormick
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D002413:Cations, Divalent; D008670:Metals; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022532:riboflavin kinase