A study of binary complexes of Escherichia coli maltodextrin phosphorylase: alpha-D-glucose 1-methylenephosphonate as a probe of pyridoxal 5'-phosphate-substrate interactions.
The glucose 1-phosphate (Glc-1-P) analog alpha-D-glucose 1-methylenephosphonate (Glc-1-MeP) inhibits competitively Escherichia coli maltodextrin phosphorylases against Glc-1-P (Ki = 0.20 mM) but also Pi (Ki = 0.36 mM). Exchange of the active site residue Glu637 to Asp by site-directed mutagenesis abolishes inhibition only in the synthesis direction (S-mode), while the degradative direction (P-mode) was not affected. Structural and conformational differences of the S-mode versus P-mode were also revealed by 31P-NMR spectroscopy by comparing chemical shifts of the cofactor pyridoxal-P in binary complexes formed either in the presence of Glc-1-MeP or of arsenate. In contrast the apparent pK of pyridoxal-P in both binary complexes was closely similar. Again, the total chemical shift of pyridoxal-P in the synthesis mode respectively degradative mode was differently affected in the binary complexes of the Glu637Asp mutant enzyme. This supports the contention that differential binding of the substrates in the synthesis or the degradative mode changes the arrangement and mutual interactions of cofactor phosphate and substrate phosphates.
Becker
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S
Theodor-Boveri-Institut für Biowissenschaften (Biozentrum), Universität Würzburg, Germany.
Schnackerz
K D
KD
Schinzel
R
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D001224:Aspartic Acid; D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000201:enzymology*; D005947:Glucose / Q000031:analogs & derivatives* / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*
D009943:Organophosphorus Compounds; D001224:Aspartic Acid; D018698:Glutamic Acid; D011732:Pyridoxal Phosphate; C079131:glucose-1-methylenephosphonate; D005964:Glucosyltransferases; C033938:maltodextrin phosphorylase; D005947:Glucose