The fast-reacting sulfhydryl group of rat muscle phosphoglycerate kinase is necessary for activity and maintenance of tertiary structure.
Phosphoglycerate kinase from rat muscle has no disulfide linkage but possesses four -SH groups--one fast reacting and three slow reacting to 5,5'-dithiobis (2-nitro-benzoate). The enzyme is inactivated by reaction of the fast-reacting -SH with iodoacetate. There is a stoichiometric relationship between modification of this group and enzyme inactivation. The reaction follows pseudo-first-order kinetics and is dependent upon the iodoacetate concentration; the value of the second-order rate constant is 2.6 M-1 min-1. Amino acid analysis of the enzyme after treatment with iodo[2-14C]acetate reveals that cysteine is the only amino acid to undergo modification. Moreover, the pK for the fast-reacting -SH has been found to be 9.1, a normal value for a cysteinyl residue. MgADP and ADP protect the enzyme against iodoacetate inactivation, whereas 3-phosphoglycerate, MgATP, ATP, or Mg2+ has no effect on the reaction. The dissociation constant (Kd) for MgADP as determined by iodoacetate inactivation kinetics is the same as the Km value, thus indicating that the fast-reacting -SH is in the MgADP binding domain of the enzyme. On storage, there is a loss of enzyme activity and a concomitant loss of fast-reacting -SH. Three protein peaks with Mr values of 76 000, 43 000 and 36 000 are obtained after Sephadex G-200 chromatography of the stored enzyme. The evidence indicates that the protein with Mr 43 000 is produced by unfolding of the native enzyme (Mr 36 000). The large protein (Mr 76 000) is a dimer resulting from formation of a disulfide linkage between the fast-reacting -SH of two molecules of Mr 43 000. The dimeric protein is catalytically inactive but can be fully reactivated by reducing agents. Physical and immunological properties of the three forms of the enzyme are also described.
D000818:Animals; D002942:Circular Dichroism; D007525:Isoelectric Focusing; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D010735:Phosphoglycerate Kinase