HISTIDINE-RESIDUE-139, HISTIDINE-RESIDUE-363 AND HISTIDINE-RESIDUE-500 ARE ESSENTIAL FOR CATALYTIC ACTIVITY OF COFACTOR-INDEPENDENT PHOSPHOGLYCEROMUTASE FROM DEVELOPING ENDOSPERM OF THE CASTOR PLANT

Cofactor-independent phosphoglyceromutase (PGM) from castor is inactiv ated by diethyl pyrocarbonate, implicating histidine residues in the c atalytic mechanism. Treatment of the inhibited enzyme with 1 M hydroxy lamine at pH 7.0 restores the enzyme activity. Spectroscopic data indi cate that the inactivation of PGM with diethyl pyrocarbonate is the re sult of formation of carbethoxyhistidine derivatives. The substrate, 3 -phosphoglycerate, substantially protects the enzyme against diethyl p yrocarbonate inactivation, indicating that the histidine residues impo rtant in catalysis are at or near the active site of the enzyme. There are 12 conserved histidine residues in all plant PGMs that have been sequenced. In the castor PGM, these conserved histidine residues were changed to either valine (H12V) or alanine (H41A, H65A, H84A, H127A, H 139A, H163A, H363A, H433A, H471A, H500A and H540A) by in vitro mutagen esis. Expression of these mutant proteins in Escherichia coli produced seven soluble mutant proteins (mutations H41A, H65A, H84A, H139A, H35 3A, H500A and H540A) and five insoluble mutant proteins (mutations H12 V, H127A, H163A, H433A and H471A). Among the seven soluble proteins, f our possessed normal PGM activity (mutations H41A, H65A, H84A and H540 A) and three (mutations H139A, H363A and H500A) had no catalytic activ ity. Along with the in-vitro-expressed wild-type enzyme, mutant enzyme s [H139A]PGM, [H363A]PGM and [H500A]PGM were purified to homogeneity. Purified wildtype PGM expressed in E. coli was active and had a K-m va lue very close to that of the enzyme purified from castor endosperm, w hile the three mutant enzymes remained inactive throughout purificatio n. Therefore, histidine residues 139, 363 and 500 appear to be essenti al for the catalytic activity of the cofactor-independent enzyme, and may be located at the active site. Hence, although the cofactor-depend ent and cofactor-independent PGMs have no homology in their primary am ino acid sequences, both enzymes appear to utilize histidine residues to mediate the transfers of proton and phospho groups in the reaction, and thus may be functionally and mechanistically convergent.