Isolation and properties of 5‐aminolevulinate synthase from the yeast Saccharomyces cerevisiae

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Abstract
5-Aminolevulinate synthase from yeast mitochondria was purified to homogeneity for the 1st time. By using affinity chromatography on agarose-hexane-CoA, gel filtration and DEAE-Sepharose chromatography, the enzyme was purified .apprx. 7000-fold with an overall yield of 40%. The specific activity of the final preparation was 39,000 nmol of 5-aminolevulinate h-1 mg-1 of protein at 30.degree. C. As judged by gel filtration, polyacrylamide gradient gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme appeared to be composed of 2 identical subunits of a relative molecular mass [MW] of 53,000. Electrophoresis of sodium-dodecyl-sulfate-solubilized yeast homogenate followed by immune replica analysis showed that the value of 53,000 is the MW of a non-degraded form. The purified enzyme had an isoelectric point of 5.3 and a pH optimum of 7.4. Pyridoxal 5''-phosphate was an essential cofactor. The enzyme activity was sensitive to thiol blocking reagents. Hemin, but not heme, inhibited the activity of the purified enzyme.