Transient-state kinetic analysis of Synechococcus glutamate 1-semialdehyde aminotransferase.
We report a transient-state kinetic analysis relating to the mechanism of glutamate 1-semialdehyde aminotransferase (GSAT). Multiple-wavelength spectral kinetic data were collected by micro-stopped-flow spectrophotometry. Time resolved spectral sketches resulting from reactions with glutamate 1-semialdehyde (GSA), 4,5-diaminovalerate (DAVA), and 5-aminolevulinate (ALA) indicated various transient chromophoric intermediates. On the basis of the generally accepted mechanism of other aminotransferases and absorbance characteristics of associated intermediates, these transient chromophores are likely associated with Schiff base formation, ketimine/aldimine tautomerization, and transimidation etc. Spectral kinetic changes associated with these putative intermediates were, in general, concentration dependent. Various experimental evidence, including reactions with the GSAT lys272ile mutant, suggested rapid equilibrium of isomeric aldimines and geminal diamines. With this and related simplifying assumptions, a minimal mechanism was derived which provided a means for transient-state spectral kinetic analysis of reactions with GSA, DAVA, and ALA, all of which lead to the formation of the same putative central enzyme complex. Resulting kinetic constants were internally consistent, in general agreement with steady-state and equilibrium data (KM, kcat, and Keq), and provided the basis for a reasonable computer simulation of the original data set (variance approximately 4 x 10(-5)). Reequilibration of enzyme intermediates following an apparent pseudoequilibrium indicated thermodynamically driven dissociation of the central aldiminic enzyme complex. This is consistent with previous observations and the minimal mechanism used in this kinetic analysis and suggests a plausible regulatory mechanism of GSAT.
Smith
M A
MA
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA.
King
P J
PJ
Grimm
B
B
D000599:Amino Acids, Diamino / Q000378:metabolism; D000622:Aminolevulinic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003198:Computer Simulation; D000458:Cyanobacteria / Q000201:enzymology*; D003959:Diamines / Q000378:metabolism; D007097:Imines / Q000378:metabolism; D019751:Intramolecular Transferases / Q000378:metabolism*; D007700:Kinetics; D008432:Mathematical Computing; D013053:Spectrophotometry
D000599:Amino Acids, Diamino; D001426:Bacterial Proteins; D003959:Diamines; D007097:Imines; D000622:Aminolevulinic Acid; C074874:4,5-diaminopentanoic acid; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase