Involvement and identification of a tryptophanyl residue at the pyruvate binding site of transcarboxylase.
Transcarboxylase (TC) from Propionibacterium shermanii consists of a central hexameric 12S subunit to which 6 outer dimeric 5S subunits are attached through 12 biotinyl 1.3S subunits. The enzyme catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate, forming oxalacetate and propionyl-CoA. The pyruvate binding site, located on the 5S subunit, was examined by monitoring the intrinsic fluorescence quenching accompanying the incremental addition of pyruvate to either TC or the 5S subunit. The quenching studies indicate that there are two binding sites for pyruvate with apparent dissociation constants of 0.23 and 1.25 mM for intact TC and of 0.18 and 1.20 mM for the outer 5S subunit. The microenvironment of the Trp(s) sensitive to pyruvate binding was analyzed by using the neutral quencher acrylamide. With TC, the fractional accessible fluorescence (fa) was 0.64, whereas a fa value of 0.56 was obtained in the presence of pyruvate. A 27% decrease in fa was observed with the outer 5S subunit in the presence of pyruvate as compared to the free 5S subunit. By labeling the outer subunit in the absence of pyruvate with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), a tryptic peptide containing DNPS-labeled Trp was isolated; the sequence was determined and identified with the amino-terminal residues 67-75 of the outer subunit that has been derived from DNA-sequencing studies. Trp-73 contained the DNPS label; its labeling was inhibited by pyruvate. A sequence comparison with other biotinyl enzymes shows that the sequence 67-75 is highly conserved.(ABSTRACT TRUNCATED AT 250 WORDS)
Kumar
G K
GK
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Haase
F C
FC
Phillips
N F
NF
Wood
H G
HG
D020106:Acrylamide; D000178:Acrylamides; D000595:Amino Acid Sequence; D001665:Binding Sites; D019878:Carboxyl and Carbamoyl Transferases; D004136:Dinitrobenzenes; D008969:Molecular Sequence Data; D011424:Propionibacterium / Q000201:enzymology; D011487:Protein Conformation; D011773:Pyruvates; D019289:Pyruvic Acid; D013050:Spectrometry, Fluorescence; D013434:Sulfenic Acids; D014166:Transferases / Q000378:metabolism*; D014364:Tryptophan
D000178:Acrylamides; D004136:Dinitrobenzenes; D011773:Pyruvates; D013434:Sulfenic Acids; D020106:Acrylamide; C004788:2,4-dinitrobenzenesulfenyl chloride; D019289:Pyruvic Acid; D014364:Tryptophan; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase