PMID:7703234
| Citation |
Carlow, DC, Smith, AA, Yang, CC, Short, SA and Wolfenden, R (1995) Major contribution of a carboxymethyl group to transition-state stabilization by cytidine deaminase: mutation and rescue. Biochemistry 34:4220-4 |
|---|---|
| Abstract |
The crystal structure of an inhibitory complex formed between Escherichia coli cytidine deaminase and the transition-state analog 3,4-dihydrouridine indicates the presence of a short H-bond between Glu-104 and the inhibitor. To test the possibility that analogous H-bonds might play a significant role in stabilizing the hydrated substrate in the transition state for deamination, we replaced Glu-104 by alanine. Compared with the wild-type enzyme, the mutant enzyme's affinities for substrate cytidine and product uridine were found to have increased, whereas kcat for deamination of cytidine had been reduced by 8 orders of magnitude. By its presence, the carboxymethyl group of Glu-104 appears to minimize the activation barrier for deamination, not only by stabilizing the altered substrate in the transition state but also by destabilizing the enzyme-substrate and enzyme-product complexes. In the presence of added formate ion, but not in the presence of bulkier carboxylic acids, the low catalytic activity of the mutant enzyme was enhanced substantially. |
| Links | |
| Keywords |
Alanine; Cytidine/metabolism; Cytidine Deaminase/chemistry; Cytidine Deaminase/genetics; Cytidine Deaminase/metabolism; Enzyme Activation/drug effects; Enzyme Stability; Escherichia coli/enzymology; Escherichia coli/genetics; Formates/pharmacology; Glutamic Acid; Hydrogen Bonding; Hydrogen-Ion Concentration; Mutagenesis, Site-Directed; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Structure-Activity Relationship; Substrate Specificity; Uridine/metabolism |
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