H2 formation from NADH or NADPH in cell lysates of C. kluyveri

Assay mixtureaAmt of H2 formed after 10 min (μmol)
NADH-regenerating system with:
    Acetyl-phosphate + CoAb2.2
    Propionyl-phosphate + CoAc0.01
    Propionyl-phosphate + CoA + acetated1.8
    Propionyl-phosphate + CoA + crotonatee0.01
    Propionyl-phosphate + CoA + vinylacetatef3.6
    Propionyl-phosphate + CoA + butyrateg0.01
NADPH-regenerating system with:
    Acetyl-phosphate + CoAb0.01
    Propionyl-phosphate + CoA + vinylacetatef0.01
  • a Assays with activity are indicated by bold type. Each 1-ml assay mixture incubated at 37°C contained 100 mM Tris-HCl (pH 7.5), 20 mM 2-mercaptoethanol, 10 μM FAD, 5 U phosphotransacetylase, the NADH-regenerating system (2.5 mM NADH, 20 mM galactose, and 1 U galactose dehydrogenase) or the NADPH-regenerating system (0.5 mM NADP+, 40 mM glucose-6-phosphate, and 1 U glucose-6-phosphate dehydrogenase), and 7 mg of cell lysate protein from which low-molecular-mass compounds had been removed by Sephadex G-25 gel filtration. The cell lysates contained hydrogenase, ferredoxin, and acetyl-CoA:butyrate CoA transferase. Where indicated, CoA (1 mM), acetyl-phosphate (25 mM), propionyl-phosphate (25 mM), acetate (50 mM), vinylacetate (50 mM), crotonate (50 mM), butyrate (50 mM), crotonyl-CoA (0.8 mM), and/or NAD+ (10 mM) were added (acyl-phosphates as potassium/lithium salts and fatty acids as potassium salts). Each reaction was started by addition of cell lysate.

  • b Acetyl-CoA formation was catalyzed by phosphotransacetylase (reaction 7).

  • c Propionyl-CoA formation was catalyzed by phosphotransacetylase (reaction 8).

  • d Acetyl-CoA formation was catalyzed by phosphotransacetylase and CoA transferase (reactions 8 and 9).

  • e The CoA transferase in the cell extract did not catalyze the formation of crotonyl-CoA from propionyl-CoA and crotonate (35, 43).

  • f Crotonyl-CoA formation via phosphotransacetylase, CoA transferase, and isomerase (reactions 8, 9, and 10).

  • g Butyryl-CoA formation via phosphotransacetylase and CoA transferase. The cell extracts did not catalyze the formation of 2-methylcaproyl-CoA from butyryl-CoA and propionyl-CoA.

  • h Crotonyl-CoA was completely consumed after 2 min.