The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies

Schizophyllum commune alpha,alpha-trehalose phosphorylase utilizes a glycosyl-transferase-like catalytic mechanism to convert its disaccharide substrate into alpha-D-glucose 1-phosphate and alpha-D-glucose. Recruitment of phosphate by the free enzyme induces alpha,alpha-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif ( where X is any amino acid) comprising key residues of its putative phosphate-binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (k(cat)/K-m = 21 000 M-1.s(-1)) was dramatic (>= 10(7)-fold) in purified Arg507 -> Ala (R507A) and Lys512 -> Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in k(cat) (Arg507) and K-m (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H2PO4-, the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with alpha, alpha-trehalose (K-i = 0.4 mu M) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (K-i = 300 mu M). Phosphate (50 mM) enhanced the basal hydrolase activity of the K512A mutant toward alpha, alpha-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively.