Major contribution of a carboxymethyl group to transition-state stabilization by cytidine deaminase: mutation and rescue.
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.
Carlow
D C
DC
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260.
Smith
A A
AA
Yang
C C
CC
Short
S A
SA
Wolfenden
R
R
D000409:Alanine; D003562:Cytidine / Q000378:metabolism; D003564:Cytidine Deaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005561:Formates / Q000494:pharmacology; D018698:Glutamic Acid; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014529:Uridine / Q000378:metabolism
D005561:Formates; D011994:Recombinant Proteins; C030544:formic acid; D018698:Glutamic Acid; D003562:Cytidine; D003564:Cytidine Deaminase; D000409:Alanine; D014529:Uridine