Characteristics of a hydrogen peroxide-forming pyruvate oxidase from Streptococcus sanguis

A cytoplasmic pyruvate oxidase was partially purified from Streptococcus sanguis ATCC 10556. The enzyme used pyruvate, inorganic phosphate and oxygen as substrates, and formed acetyl phosphate and hydrogen peroxide. In this reaction carbon dioxide can also be expected, but this product was not looked for. The enzyme was dependent on thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD) and magnesium ions for activity. Its relative molecular weight (Afr) was 260,000 as calculated from its migration distance in polyacrylamide gradient gel. The enzyme was hysteretic and its activity was not influenced by various low molecular-weight substances known to be present in the cytoplasm. The pH optimum of the enzyme was 6.7 to 7.5 and its activity was not inhibited by 1 mM hydrogen peroxide or iodoacetamide, or by 10 mM potassium cyanide, sodium azide, iodoacetate, sodium fluoride, zinc chloride, EDTA, or dithiothreitol.