Arginine-239 in the beta subunit is at or near the active site of bovine pyruvate dehydrogenase.
We have modified bovine pyruvate dehydrogenase (E1), the first catalytic component of the pyruvate dehydrogenase complex, with pyreneglyoxal. Treatment of E1 with pyreneglyoxal resulted in the loss of enzyme activity. Pyruvate plus thiamin pyrophosphate (TPP) afforded approximately 80% protection against this inactivation and protected two arginine residues per mol of E1 tetramer (alpha 2 beta 2) from modification. Circular dichroism spectral analysis indicated absence of any gross structural changes in the enzyme as a result of modification. Comparison of the peptide maps, monitored at 345 nm of unprotected and pyruvate plus TPP protected E1s after V8 digestion revealed that a peptide in the protected enzyme was labeled by pyreneglyoxal to a lesser extent than its counterpart in the unprotected enzyme. Sequence analysis of the peptide demonstrated that it corresponded precisely to amino-acid residues 235 to 246 in the human E1 beta sequence, with arginine residues at positions 239 and 242. Since Arg-239 is conserved in the beta-subunit of all presently known sequences of the pyruvate dehydrogenase complex and branched-chain alpha-keto acid dehydrogenase complex, it is strongly suggested that Arg-239 in the human E1 beta sequence is at or near the active site of bovine E1.
Eswaran
D
D
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Ali
M S
MS
Shenoy
B C
BC
Korotchkina
L G
LG
Roche
T E
TE
Patel
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000032:analysis*; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D010450:Endopeptidases; D006037:Glyoxal / Q000031:analogs & derivatives; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011721:Pyrenes; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry*
D010455:Peptides; D011721:Pyrenes; D011768:Pyruvate Dehydrogenase Complex; C066961:pyrene glyoxal; D006037:Glyoxal; D001120:Arginine; D010450:Endopeptidases