IDENTIFICATION OF A SITE IN THE PHOSPHOCARRIER PROTEIN, HPR, WHICH INFLUENCES ITS INTERACTIONS WITH SUGAR PERMEASES OF THE BACTERIAL PHOSPHOTRANSFERASE SYSTEM - KINETIC ANALYSES EMPLOYING SITE-SPECIFIC MUTANTS

The permeases of the Escherichia coli phosphoenolpyruvate:sugar phosph otransferase system (PTS), the sugar-specific enzymes II, are energize d by sequential phosphoryl transfer from phosphoenolpyruvate to (i) en zyme I, (ii) the phosphocarrier protein HPr, (iii) the enzyme IIA doma ins of the permeases, and (iv) the enzyme IIBC domains of the permease s which transport and phosphorylate their sugar substrates. A number o f site-specific mutants of HPr were examined by using kinetic approach es. Most of the mutations exerted minimal effects on the kinetic param eters characterizing reactions involving phosphoryl transfer from phos pho-HPr to various sugars. However, when the well-conserved aspartyl 6 9 residue in HPr was changed to a glutamyl residue, the affinities for phospho-HPr of the enzymes II specific for mannitol, N-acetylglucosam ine, and beta-glucosides decreased markedly without changing the maxim al reaction rates. The same mutation reduced the spontaneous rate of p hosphohistidyl HPr hydrolysis but did not appear to alter the rate of phosphoryl transfer from phospho-enzyme I to HPr. When the adjacent gl utamyl residue 70 in HPr was changed to a lysyl residue, the V-max val ues of the reactions catalyzed by the enzymes II were reduced, but the K-m values remained unaltered. Changing this residue to alanine exert ed little effect. Site-specific alterations in the C terminus of the b eta-glucoside enzyme II which reduced the maximal reaction rate of pho sphoryl transfer about 20-fold did not alter the relative kinetic para meters because of the aforementioned mutations in HPr. Published three -dimensional structural analyses of HPr and the complex of HPr with th e glucose-specific enzyme IIA (IIA(Glc)) (homologous to the beta-gluco side and N-acetylglucosamine enzyme IIA domains) have revealed that re sidues 69 and 70 in HPr are distant from the active phosphorylation si te and the IIA(Glc) binding interface in HPr. The results reported the refore suggest that residues D-69 and E-70 in HPr play important roles in controlling conformational aspects of HPr that influence (i) autop hosphohydrolysis, (ii) the interaction of this protein with the sugar permeases of the bacterial phosphotransferase system, and (iii) cataly sis of phosphoryl transfer to the IIA domains in these permeases.