ARCHAEAL PHOSPHOPROTEINS - IDENTIFICATION OF A HEXOSEPHOSPHATE MUTASEAND THE ALPHA-SUBUNIT OF SUCCINYL-COA SYNTHETASE IN THE EXTREME ACIDOTHERMOPHILE SULFOLOBUS-SOLFATARICUS

When soluble extracts from the extreme acidophilic archaeon Sulfolobus solfataricus were incubated with [gamma-P-32]ATP, several radiolabele d polypeptides were observed following SDS-PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa Phosphoamino acid analysis revealed that all of the pro teins contained phosphoserine, with the exception of the 35-kDa one, w hose protein-phosphate linkage proved labile to strong acid. The obser ved pattern of phosphorylation was influenced by the identity of the d ivalent metal ion cofactor used, Mg2+ versus Mn2+, and the choice of i ncubation temperature. The 35- and 50-kDa phosphoproteins were purifie d and their amino-terminal sequences determined. The former polypeptid e's amino-terminal sequence closely matched a conserved portion of the Lu-subunit of succinyl-CoA synthetase, which forms an acid-labile pho sphohistidyl enzyme intermediate during its catalytic cycle. This iden tification was confirmed by the ability of succinate or ADP to specifi cally remove the radiolabel. The 50-kDa polypeptide's sequence contain ed a heptapeptide motif, Phe/Pro-Gly-Thr-Asp/Ser-Gly-Val/Leu-Arg, foun d in a similar position in several hexosephosphate mutases. The cataly tic mechanism of these mutases involves formation of a phosphoseryl en zyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [P-32]glucose 6-phosphate to donate its phosphoryl group t o the protein.