Abstract:
:Comparisons of catalytic mechanisms have not previously been performed for homologous enzymes from hyperthermophilic and mesophilic sources. Here, the beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was recombinantly produced in Escherichia coli and shown to have biophyscial and biochemical properties identical to those of the wild-type enzyme. Moreover, the recombinant enzyme was subjected to a detailed kinetic investigation at 95 degreesC to compare its catalytic mechanism to that determined at 37 degreesC for the beta-glucosidase (abg) from the mesophilic bacterium, Agrobacterium faecalis [Kempton, J., and Withers, S. G. (1992) Biochemistry 31, 9961]. These enzymes have amino acid sequences that are 33% identical and have been classified as family 1 glycosyl hydrolases on the basis of amino acid sequence similarities. Both enzymes have similar broad specificities for both sugar and aglycone moieties and exhibit nearly identical pH dependences for their kinetic parameters with several different substrates. Bronsted plots were constructed for bgl at several temperatures using a series of aryl glucoside substrates. These plots were concave downward at all temperatures, indicating that bgl utilized a two-step mechanism similar to that of abg and that the rate-limiting step in this mechanism did not change with temperature for any given aryl glucoside. The Bronsted coefficient for bgl at 95 degreesC (beta1g = -0.7) was identical to that for abg at 37 degreesC and implies that these enzymes utilize nearly identical transition states, at least in regard to charge accumulation on the departing glycosidic oxygen. In addition, a high correlation coefficient (rho = 0.97) for the linear free energy relationship between these two enzymes and similar inhibition constants for these two enzymes with several ground state and transition state analogue inhibitors further indicate that these enzymes stabilize similar transition states. The mechanistic similarities between these two enzymes are noteworthy in light of the large difference in their temperature optima. This suggests that, in the presumed evolution that occurred between the hyperthermophilic archaeal enzyme and the mesophilic bacterial enzyme, structural modifications must have been selected which maintained the integrity of the active site structure and, therefore, the specificity of transition state interactions, while adapting the overall protein structure to permit function at the appropriate temperature.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Bauer MW,Kelly RMdoi
10.1021/bi9814944subject
Has Abstractpub_date
1998-12-08 00:00:00pages
17170-8issue
49eissn
0006-2960issn
1520-4995pii
bi9814944journal_volume
37pub_type
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