Site-directed mutagenesis and the role of the oxyanion hole in subtilisin.

Abstract

Oligonucleotide-directed mutagenesis was used to investigate the nature of transition state stabilization in the catalytic mechanism of the serine protease, subtflisin BPN'. The gene for this extracellular enzyme from Bacillus amyloliquefaciens has been cloned and expressed in Bacillus subtilis. In the transition state complex, the carbonyl group of the peptide bond to be hydrolyzed is believed to adopt a tetrahedral configuration rather than the ground-state planar configuration. Crystallographic studies suggest that stabilization of this activated complex is accomplished in part through the donation of a hydrogen bond from the amide side group of Asn-155 to the carbonyl oxygen of the peptide substrate. To specifically test this hypothesis, leucine was introduced at position 155. Leucine is isosteric with asparagine but is incapable of donating a hydrogen bond to the tetrahedral intermediate. The variant was found to have an unaltered K. but a greatly reduced catalytic rate constant, kcat, (factor of 200-300 smaller) when assayed with a peptide substrate. These kinetic results are consistent with the Asn-155 mediating stabilization of the activated complex and lend further experimental support for the transition-state stabilization hypothesis of enzyme catalysis.

Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution

“…Finally, the success of these experiments shows that in vitro translation can be used to generate sufficient quantities of backbone amide to ester substitutions to test the importance of their interactions with substrates/intermediates/transition states interactions in other enzymes (e.g., serine proteases, haloalkane dehalogenases, and enoyl-CoA hydratase) (2,20,21).…”

“…Such an interaction can contribute to catalysis by increasing in strength as the basicity/proton affinity of the acceptor is increased as the reaction coordinate is traversed (1). Perhaps the best known example of such an interaction is the oxyanion hole in serine proteases, in which the substrate peptide carbonyl group is converted to an anionic tetrahedral intermediate (2). However, the importance of the presumed enhanced hydrogen-bonding interaction between the oxyanion hole and the substrate/intermediate has not been directly evaluated because of the difficulty in mutating the backbone amide group donor and/or the substrate acceptor.…”

Serine proteases: An ab initio molecular dynamics study

“…Stabilization of the intermediate is achieved by formation of two H-bonds with the amide groups of Ser195 and Gly193 (mammalian isoenzymes 4 ) or with the amide groups of Ser195 and the side chain of Asn155 (bacterial isoenzymes 14 ).…”

Protein engineering. The design, synthesis and characterization of factitious proteins

“…1 (Robertus et al, 1972;Kossiakof & Spencer, 1981). The studies of and Bryan et al (1986) give useful estimates of the quantitative importance of hydrogen bonds to transition state stabilization, taking an approach similar to that by Fersht and his colleagues for tyrosyl-tRNA synthetase (see below). The calculated differences in free energy differences between the transition state and the ground state from:…”

“…Such information is not yet available for many mutant enzymes, and subtilisin is no exception in this respect, but for the substrate used in the above study it is known that the acylation step is rate-limiting and that the ratio of rate constants for acylation and de-acylation is comfortably high (33) for the wild type enzyme. Since Asn-155--Leu is a nearly isosteric change to a residue which cannot form hydrogen bonds, it is comforting that the calculated AAG* value of 15 kJ -mol-1 (Bryan et al, 1986) (Russell et al, 1987) have emphasized the importance of environmental factors near the catalytic centre and, in particular, the likely importance of electrostatic effects and large changes in the pH dependence which may accompany the removal by mutation of negative charges at surface residues up to 15 nm from the active site. In the case of subtilisin the apparent pK of His-64 can be reduced by 0.4 units by Asp-99-*Ser or Glu-156-+Ser, the functional changes in each case being masked, as predicted, by high ionic strength.…”