PYRIDOXAL 5'-PHOSPHATE AS A P-31 REPORTER OBSERVING FUNCTIONAL-CHANGES IN THE ACTIVE-SITE OF ESCHERICHIA-COLI MALTODEXTRIN PHOSPHORYLASE AFTER SITE-DIRECTED MUTAGENESIS

Changes in the active site of Escherichia coli maltodextrin phosphorylase created by substituting residues Lys533, Arg534, Tyr538, and Glu637 were monitored in the absence and presence of arsenate as substrate analogue using pyridoxal-P as P-31 NMR reporter. The chemical shift of the cofactor phosphate group of wild-type E. coli phosphorylase is pH dependent with an apparent pK of 5.6 and limiting delta-values of 0.71 and 3.6 ppm for the low- and high-pH values, respectively. The apparent pK value of 5.6 indicates that the phosphate group of the cofactor is in hydrogen bond linkage to Lys533. In all mutant enzymes in which the enzymatic activity was significantly reduced, effects on the P-31 chemical shift pattern of pyridoxal-P were observed. The K533S, R534Q, E637D, and E637Q mutant enzymes show 0.6, 0.01, 0.2, or 0.1% residual activity, and the apparent pK values of the cofactor phosphate transition of E637D and E637Q mutant enzymes are altered, The Y538F mutant enzyme is a remarkable exception, displaying 12% activity and an environment of the cofactor quite similar to that in wild-type enzyme. This finding suggests that Tyr538, although involved in substrate binding and specificity, is not functionally essential. One crucial aspect of catalysis is the close contact of the phosphates of pyridoxal-P and of substrate rendered by a cluster of positively charged amino acids, Lys533, Lys539, and Arg534. The similar apparent pK values of wild-type and K533S mutant phosphorylase suggest that the cofactor phosphate and the hydroxyl group of Ser533 are linked by a hydrogen bond. Model building studies using the R-state of muscle phosphorylase predict that such a hydrogen bond distance can only be provided by a movement of the cofactor pyridine ring including a distortion of O-P-O bond angles of the phosphate group which in turn causes the observed reduction in total P-31 chemical shift. Since residues Arg534 and Glu637 are not in close contact to the cofactor phosphate, the interpretation of the P-31 NMR data must include the participation of other neighboring amino acid residues. The P-31 NMR spectroscopy implies differences in binary complexes of E. coli mutant phosphorylases with arsenate when compared to that of native enzyme.