Biomedical subjects
Publications and source records attributed to S Braxton.
In the past year, microarray technologies have moved beyond the proof-of-principle stage. Microarrays are now being used for genome-wide expression monitoring, large-scale polymorphism screening and mapping, and for the evaluation of drug candidates.
A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis. These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase. Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA. However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease.
Stabilization of an oxyanion transition state is important to catalysis of peptide bond hydrolysis in all proteases. For subtilisin BPN', a bacterial serine protease, structural data suggest that two hydrogen bonds stabilize the tetrahedral-like oxyanion intermediate: one from the main chain NH of Ser221 and another from the side chain NH2 of Asn155. Molecular dynamic studies (Rao, S., N., Singh, U., C. Bush, P. A., and Kollman, P. A. (1987) Nature 328, 551-554) have indicated the gamma-hydroxyl of Thr220 may be a third hydrogen bond donor even though it is 4A away in the static x-ray structure. We have probed the role of Thr220 by replacing it with serine, cysteine, valine, or alanine by site-directed mutagenesis. These substitutions were intended to alter the size and hydrogen bonding ability of residue 220. Removal of the gamma-hydroxyl group reduced the transition state stabilization energy (delta delta GT) by 1.8-2.1 kcal/mol depending upon the substitution. By comparison, removal of the gamma-methyl group in the Thr220 to serine mutation only decreased delta GT by 0.5 kcal/mol. The gamma-hydroxyl of Thr220 is most important for catalysis, not substrate binding, because virtually all of the effects were on kcat and not KM. The role of the Thr220 hydroxyl is functionally independent from the amide NH2 of Asn155 because the free energy effects of double alanine mutants at these two positions are additive. These data indicate that a distal hydrogen bond donor, namely the hydroxyl of Thr220, plays a functionally important role in stabilizing the oxyanion transition state in subtilisin which is independent of Asn155.