TY - JOUR
T1 - Molecular Properties of Pyruvate Formate-Lyase Activating Enzyme
AU - Wong, Kenny K.
AU - Murray, Brion W.
AU - Lewisch, Sandra A.
AU - Baxter, Michael K.
AU - Ridky, Todd W.
AU - Ulissi-DeMario, Lucy
AU - Kozarich, John W.
PY - 1993
Y1 - 1993
N2 - 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 µM, 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. Protein radical formation requires Fe(II). Several other divalent metal ions were found to be inhibitory to protein radical formation with Cu(II), Zn(II), and Cd(II) exhibiting the most potent inhibition. The putative Fe(II) binding domain shares homologies with two other proteins found in the protein databases.
AB - 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 µM, 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. Protein radical formation requires Fe(II). Several other divalent metal ions were found to be inhibitory to protein radical formation with Cu(II), Zn(II), and Cd(II) exhibiting the most potent inhibition. The putative Fe(II) binding domain shares homologies with two other proteins found in the protein databases.
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U2 - 10.1021/bi00214a005
DO - 10.1021/bi00214a005
M3 - Article
C2 - 8260492
AN - SCOPUS:0027729952
SN - 0006-2960
VL - 32
SP - 14102
EP - 14110
JO - Biochemistry
JF - Biochemistry
IS - 51
ER -