Enzymatic conversion of glutamate to delta-aminolevulinate in soluble extracts of the unicellular green alga, Chlorella vulgaris.
Cell-free preparations from the unicellular green alga, Chlorella vulgaris, catalyze the conversion of glutamate to delta-aminolevulinate, which is the first committed step in heme and chlorophyll biosynthesis. Most activity remains in the supernatant fraction after centrifugation at 264,000g. Additional activity can be solubilized from the high-speed pellet by treatment with 0.5 M NaCl. After gel filtration through Sephadex G-25, the reaction catalyzed by the high-speed supernatant requires glutamate, ATP, Mg2+, and NADPH. Boiled extract is inactive. The pH optimum is between 7.8 and 7.9 and the temperature optimum is 30 degrees C. Concentrations required for half-maximal activity are 0.05 mM glutamate, 0.4 mM ATP, 6 mM MgCl2, and 0.4 mM NADPH or 0.7 mM NADH. The reaction requires no additional amino donor. Involvement of pyridoxal phosphate in the catalytic mechanism is suggested by sensitivity to pyridoxal antagonists; 50% inhibition is achieved with 5 microM gabaculine or 0.4 mM aminooxyacetate. Involvement of two or more enzymes is suggested by the nonlinear reaction rate dependence on protein concentration. Evidence for the involvement of an activated glutamate intermediate was obtained by product formation after sequential addition and removal of substrates, and by inhibition (80%) with 1 mM hydroxylamine. Protoheme inhibits the activity by 50% at 1.2 microM. Preincubation of the extract with ATP causes stimulation and/or stabilization of the activity compared to preincubation without ATP or no preincubation. In preparations obtained from C. vulgaris strain C-10, which requires light for greening, dark-grown cells yield one-third as much activity as 4-h-greened cells.
D000255:Adenosine Triphosphate / Q000502:physiology; D000622:Aminolevulinic Acid / Q000096:biosynthesis*; D001665:Binding Sites; D002384:Catalysis; D002708:Chlorella / Q000201:enzymology* / Q000648:ultrastructure; D005971:Glutamates / Q000378:metabolism*; D006418:Heme / Q000494:pharmacology; D007982:Levulinic Acids / Q000096:biosynthesis*; D009249:NADP / Q000502:physiology; D009711:Nucleotides / Q000502:physiology; D012996:Solutions; D013696:Temperature
D005971:Glutamates; D007982:Levulinic Acids; D009711:Nucleotides; D012996:Solutions; D006418:Heme; D009249:NADP; D000622:Aminolevulinic Acid; D000255:Adenosine Triphosphate