Transient kinetic studies support refinements to the chemical and kinetic mechanisms of aminolevulinate synthase.
5-Aminolevulinate synthase catalyzes the pyridoxal 5'-phosphate-dependent condensation of glycine and succinyl-CoA to produce carbon dioxide, CoA, and 5-aminolevulinate, in a reaction cycle involving the mechanistically unusual successive cleavage of two amino acid substrate alpha-carbon bonds. Single and multiple turnover rapid scanning stopped-flow experiments have been conducted from pH 6.8-9.2 and 5-35 degrees C, and the results, interpreted within the framework of the recently solved crystal structures, allow refined characterization of the central kinetic and chemical steps of the reaction cycle. Quinonoid intermediate formation occurs with an apparent pK(a) of 7.7 +/- 0.1, which is assigned to His-207 acid-catalyzed decarboxylation of the alpha-amino-beta-ketoadipate intermediate to form an enol that is in rapid equilibrium with the 5-aminolevulinate-bound quinonoid species. Quinonoid intermediate decay occurs in two kinetic steps, the first of which is acid-catalyzed with a pK(a) of 8.1 +/- 0.1, and is assigned to protonation of the enol by Lys-313 to generate the product-bound external aldimine. The second step of quinonoid decay defines k(cat) and is relatively pH-independent and is assigned to opening of the active site loop to allow ALA dissociation. The data support important refinements to both the chemical and kinetic mechanisms and indicate that 5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathan's hypothesis.
Hunter
Gregory A
GA
Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida 33612-4799, USA. ghunter@health.usf.edu
Zhang
Junshun
J
Ferreira
Gloria C
GC
D000624:5-Aminolevulinate Synthetase / Q000737:chemistry*; D000622:Aminolevulinic Acid / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002245:Carbon Dioxide / Q000737:chemistry; D002384:Catalysis; D003065:Coenzyme A / Q000737:chemistry; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D013237:Stereoisomerism; D013696:Temperature
D002245:Carbon Dioxide; D000622:Aminolevulinic Acid; D000624:5-Aminolevulinate Synthetase; D008239:Lysine; D003065:Coenzyme A