Molecular properties of pyruvate formate-lyase activating enzyme.
Pyruvate formate-lyase is a radical-containing enzyme that catalyzes the nonoxidative cleavage of pyruvate via a postulated homolytic mechanism. The formation of this enzymic radical in vitro requires an activating system composed of PFL-activating enzyme, S-adenosylmethionine, ferrous ion, a reduced flavin, DTT, and pyruvate as an allosteric effector. The need for large quantities of PFL-activating enzyme for biochemical and biophysical studies on the mechanism of protein radical formation has prompted us to clone the act gene and overexpress the gene product in Escherichia coli. Using PCR technology, the act gene was isolated and subcloned into various expression vectors. The overexpression of the protein was as high as 30-50% of the total cellular protein. However, the majority of the protein resided in the form of insoluble inclusion bodies. A procedure was developed to denature and isolate the inclusion bodies followed by refolding under anaerobic conditions. This purification method affords 5 mg of purified protein from 1 g of cells. Biochemical characterization demonstrated that the enzyme can bind one Fe(II) per protein monomer, and the protein did not exhibit any visible chromophore as previously observed. Co(II) and Cu(II) can be reconstituted into the protein with similar stoichiometries. Kinetic studies showed that the rate of radical formation was independent of ionic strength and the Km's for SAM and inactive PFL were determined to be 2.8 and 1.2 microM, respectively. Fluorescent binding data revealed that the Kd for SAM binding to the activating enzyme alone was comparable to the Km for SAM in the PFL activation indicating that the binding site for SAM resides on AE.(ABSTRACT TRUNCATED AT 250 WORDS)
Wong
K K
KK
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Murray
B W
BW
Lewisch
S A
SA
Baxter
M K
MK
Ridky
T W
TW
Ulissi-DeMario
L
L
Kozarich
J W
JW
D000123:Acetyltransferases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D004798:Enzymes / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012436:S-Adenosylmethionine / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004798:Enzymes; D008667:Metalloproteins; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D000123:Acetyltransferases; C040285:pyruvate formate-lyase activating enzyme; C022560:formate C-acetyltransferase