The roles of His-167 and His-275 in the reaction catalyzed by glutamate decarboxylase from Escherichia coli.
Two histidine residues in glutamate decarboxylase from Escherichia coli, potential participants in catalysis because they are conserved among amino acid decarboxylases and because they are at the active site in the homologous enzyme ornithine decarboxylase, were mutated. His-275 is shown to bind the cofactor pyridoxal 5'-phosphate but not to contribute directly to catalysis. The H275N enzyme was unable to bind the cofactor whereas the H275Q mutant contained 50% of the normal complement of cofactor and its specific activity (expressed per mole of cofactor) was 70% of that of the wild-type enzyme. The H167N mutant bound the cofactor tightly, its specific activity was approximately half that of the wild-type enzyme and experiments in D2O showed that it catalyzed replacement of the carboxyl group with retention of configuration as does the wild-type enzyme. Comparison of reaction profiles by observing changes in the absorbance of the cofactor after stopped-flow mixing, revealed that a slow reaction, in which approximately one-third of the wild-type enzyme is converted to an unreactive complex during catalysis, does not occur with the H167N mutant enzyme. This reaction is attributed to a substrate-induced conformational change, a proposal that is supported by differential scanning calorimetry.
Tramonti
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A
Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università La Sapienza, Roma, Italy.
De Biase
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Giartosio
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Bossa
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John
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RA
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002942:Circular Dichroism; D003903:Deuterium / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment; D013054:Spectrophotometry, Atomic
D006639:Histidine; D003903:Deuterium; D005968:Glutamate Decarboxylase; D009955:Ornithine Decarboxylase