Involvement of an arginyl residue in the nucleotide-binding site of Ca(2+)-ATPase from sarcoplasmic reticulum as seen by reaction with phenylglyoxal.
1. Chemical modification of the Ca(2+)-ATPase with phenylglyoxal, as a modifier of arginine residues, leads to an almost total loss of the ATPase activity. The presence of nucleotides in the reaction medium protects against the binding of 18 nmol of phenylglyoxal/mg of protein and this reduction in the binding of phenylglyoxal is accompanied by a substantial retention of ATPase activity. The incorporation of phenylglyoxal to the protein alters neither calcium binding nor phosphorylation from inorganic phosphate. Nevertheless the binding of nucleotides is dramatically inhibited and, consequently, so is phosphorylation from ATP. Fluorescein 5'-isothiocyanate labelling of the phenylglyoxal-modified ATPase is not affected but, on the other hand, phenylglyoxal is not able to modify the fluorescein 5'-isothiocyanate-prelabelled ATPase. The way in which ATPase inhibition depends on the presence of phenylglyoxal indicates that this process occurs in a pseudo-first-order reaction. However, the dependence of the apparent first-order rate constant on phenylglyoxal concentration appears to be more complex and an inhibition mechanism of two steps, with phenylglyoxal binding, has to be taken into account. 2. We have found that phenylglyoxal labels both A and B tryptic fragments, but only B fragment labelling is prevented by ATP. The sequencing of peptides from mild acid hydrolysis of phenylglyoxal-labelled ATPase shows that phenylglyoxal is located in the Ala506-Gly595 peptide that is a part of the B fragment. 3. We conclude that phenylglyoxal inactivates the calcium pump in a two-step mechanism in which the second step is irreversible. Phenylglyoxal labels an arginyl residue in the Ala506-Gly595 peptide that can be protected by the binding of ATP to its site.
Corbalán-García
S
S
Departamento de Bioquímica y Biología Molecular A, Edificio de Veterinaria, Universidad de Murcia, Spain.
Teruel
J A
JA
Gómez-Fernández
J C
JC
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010658:Phenylglyoxal / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D017421:Sequence Analysis
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D001120:Arginine; D000252:Calcium-Transporting ATPases; D010658:Phenylglyoxal; D002118:Calcium