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.
A high-D-xylulose mixture (D-xylose-D-xylulose = 33:67) was prepared from the cold ethanol extract of preisomerized D-xylose solution (D-xylose-D-xylulose = 77: 23). Fusarium oxysporum f. sp. lini and Aspergillus niger were demonstrated to preferentially utilize D-xylose in the mixture of D-xylose and D-xylulose. Chromatographically pure D-xylulose was thus obtained in 90% yield. A high-D-xylulose mixture was also incubated with Rhodotorula toruloides, Klebsiellapneumoniae, Candida utilis, or Mucor rouxii. D-Xylose and D-xylulose were simultaneously consumed. When borate was added to the mixture, a D-xylulose-borate complex was formed, and it could be used to protect D-xylulose from being utilized.
Four yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilus, and Rhodotorula toruloides) were tested for their ability to grow and consume D-glucose, D-xylose, D-xylulose, and D-xylitol. Sequential utilization of substrates was observed when D-glucose as mixed with D-xylulose as the carbon source. Catabolite inhibition was tentatively concluded to be responsible for this regulatory mechanism. D-Glucose was also found to inhibit the utilization of D-xylose and D-xylitol in C. utilus and R. toruloides. D-Xylose, D-xylitol, and D-xylulose were consumed simultaneously by R. toruloides and C. utilus.
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