Catalytic mechanism of alpha-retaining glucosyl transfer by Corynebacterium callunae starch phosphorylase: the role of histidine-334 examined through kinetic characterization of site-directed mutants.
Purified site-directed mutants of Corynebacterium callunae starch phosphorylase in which His-334 was replaced by an alanine, glutamine or asparagine residue were characterized by steady-state kinetic analysis of enzymic glycosyl transfer to and from phosphate and studies of ligand binding to the active site. Compared with wild-type, the catalytic efficiencies for phosphorolysis of starch at 30 degrees C and pH 7.0 decreased approx. 150- and 50-fold in H334Q (His334-->Gln) and H334N mutants, and that of H334A was unchanged. In the direction of alpha-glucan synthesis, selectivity for the reaction with G1P (alpha-D-glucose 1-phosphate) compared with the selectivity for reaction with alpha-D-xylose 1-phosphate decreased from a wild-type value of approximately 20000 to 2600 and 100 in H334N and H334Q respectively. Binding of G1P to the free enzyme was weakened between 10-fold (H334N, H334Q) and 50-fold (H334A) in the mutants, whereas binding to the complex of enzyme and alpha-glucan was not affected. Quenching of fluorescence of the pyridoxal 5'-phosphate cofactor was used to examine interactions of the inhibitor GL (D-gluconic acid 1,5-lactone) with wild-type and mutant enzymes in transient and steady-state experiments. GL binding to the free enzyme and the enzyme-phosphate complex occurred in a single step. The 50-fold higher constant (K(d)) for GL dissociation from H334Q bound to phosphate resulted from an increased off-rate for the ligand in the mutant, compared with wild-type. A log-log correlation of catalytic-centre activity for phosphorolysis of starch with a reciprocal K(d) value established a linear free-energy relationship (slope=1.19+/-0.07; r2=0.991) across the series of wild-type and mutant enzymes. It reveals that GL in combination with phosphate has properties of a transition state analogue and that the His-334 side chain has a role in selectively stabilizing the transition state of the reaction.
Schwarz
Alexandra
A
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Pierfederici
Francesco Maria
FM
Nidetzky
Bernd
B
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D003352:Corynebacterium / Q000201:enzymology*; D005942:Gluconates; D005947:Glucose / Q000378:metabolism*; D006639:Histidine; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D024941:Starch Phosphorylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005942:Gluconates; D006639:Histidine; D024941:Starch Phosphorylase; D005947:Glucose; C030691:gluconic acid