Stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate in aspartate aminotransferase. Activators and inhibitors.
Measurement of the stereospecific release of the pro-S proton from C-4' of enzyme-bound pyridoxamine 5'-phosphate provides an experimental means to probe parts of the active site of aspartate aminotransferase independently of substrate turnover (Tobler, H. P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). The release of pro-S 3H from enzyme-bound [3H]pyridoxamine 5'-phosphate is 30,000 times faster than from free coenzyme. Enzyme-bound [3H]pyridoxine 5'-phosphate is not detritiated suggesting an essential role of the 4'-amino group. Formation of the unproductive complex of the [3H]pyridoxamine 5'-phosphate-enzyme with aspartate or glutamate results in a 400-fold acceleration of 3H release. In contrast, addition of borohydride or cyanoborohydride immediately stops 3H release. Experiments with a fluorescent reporter group and with differential chemical modifications indicate that the activating effect of aspartate on the release of 3H is accompanied by a shift of the so-called open/closed conformational equilibrium of the enzyme (Kirsch, J.F., Eichele, G., Ford, G. C., Vincent, M.G., Jansonius, J.N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525) toward the closed conformation; the inhibiting effect of borohydride and cyanoborohydride appears to be accompanied by a shift toward the open conformation. Apparently, at least part of the catalytic apparatus of aspartate aminotransferase becomes fully operative only in the closed conformation of the enzyme.
D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D001894:Borohydrides; D002645:Chickens; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D011487:Protein Conformation; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D001894:Borohydrides; D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; C010627:pyridoxamine phosphate