Site-directed mutagenesis of @entity526 XXXX transcarbamoylase: role of @entity369 -57 in substrate binding and catalysis.

@entity7792 @entity12116 @entity2684 @entity2228 @entity535 @entity68881 @entity4022 @entity7222 @entity534 @entity1391 @entity21100 @entity10608 @entity369 @entity526
In the @entity21100 -transfer reaction catalyzed by @entity7222 transcarbamoylase, an @entity369 residue in the active site of the @entity526 enzyme has been suggested to bind the @entity1391 moiety of the substrate @entity12116 . With the application of site-specific mutagenesis, the most likely @entity369 residue among three candidates at the binding site of @entity12116 , @entity534 -57, has been replaced with a @entity2228 . The resultant @entity4022 -57 mutant enzyme is drastically inefficient in catalysis. In the synthesis of @entity7792 from @entity12116 and @entity10608 with the release of inorganic @entity1391 , the turnover rate of the mutant is 21,000-fold lower than that of the wild type. However, the mutation of @entity534 -57 affects only moderately the binding of @entity12116 ; the dissociation constant of this substrate, measured under steady-state turnover condition, is increased from 0.046 to 3.2 mM by the mutation. On the other hand, @entity7222 binding is substantially affected as estimated by the change in the dissociation constant of its analogue @entity68881 . The dissociation constant of @entity68881 increases about 500-fold from 54 microM for the wild type to 25 mM for the mutant. Since @entity534 -57 is expected to be distal from the @entity7222 site and the @entity535 (both @entity7222 and @entity2684 ) binds only after @entity12116 in the wild-type reaction, the poor norvaline affinity to the mutant suggests that @entity534 -57 is involved in interactions essential for productive addition of the @entity535 . This interpretation is supported by difference ultraviolet absorption spectra which show that the conformational changes induced in the wild type by @entity12116 upon binding are absent in the mutant. Furthermore, steady-state kinetic data reveal that the ordered binding mechanism of the wild-type enzyme is transformed into a random binding mechanism in the mutant. Thus, the presence of @entity12116 in the mutant active site is no longer a requisite for @entity7222 binding. In the 5-50 degrees C temperature range, transcarbamoylation catalyzed by either the wild type or the mutant observes the Arrhenius rate law with almost identical enthalpies of activation, 11 and 10 kcal/mol, respectively. The entropy of activation is -5.5 eu for the wild-type reaction and -29 eu for the mutant reaction, accounting for a loss of 6-7 kcal/mol in the rate-determining step of the enzymic reaction.(ABSTRACT TRUNCATED AT 400 WORDS)
