tailieunhanh - ENZYME KINETICS A MODERN APPROACH – PART 10

Cả hai đột biến cho thấy một tỷ lệ ngừng hoạt động chậm hơn khoảng 1,5 lần hơn so với các loại hoang dã, trong khi đột biến N-Frag 5,8 lần chậm hơn. Ngoài ra, ở pH , các hoạt động của cả hai N-Frag (A) và N-Frag (B) đột biến đã được dập tắt chậm hơn so với hoang dại, nhưng nhanh hơn so với đột biến N-Frag. | 210 ENZYME KINETIC DATA IN PROTEINS STRUCTURE-FUNCTION STUDIES Fig. . Both mutants showed an inactivation rate about times slower than that of the wild-type while the N-frag mutant was times slower. Also at pH the activities of both N-frag A and N-frag B mutants were quenched more slowly than the wild-type but faster than the N-frag mutant. After 5 minutes N-frag A N-frag B N-frag and wild-type had 0 3 27 and 0 of the original activity in the absence of glycerol and sucrose while 45 46 0 and 72 of the original activities remained in the presence of glycerol and sucrose respectively. Molecular minimization using molecular modeling showed that these mutations did not contribute to the internal interactions Fig. . The only major difference between the wild-type and N-frag mutant was the addition of a hydrogen bond between serine 2-glycine oxygen and leucine-167 nitrogen in the glycine 2 serine mutation. The kinetics of the N-frag A mutant however showed that this addition was insufficient to stabilize the protein entirely. It was thus concluded that each of the five replacements by themselves were not critical but helped to stabilize the enzyme synergistically. Since each of the mutations above in itself was not critical to stabilization the mechanism of stability was still in question. We therefore suggested two possibilities of how the mutations stabilized pepsin 1 the release of N-terminus portion is prevented with these mutations or 2 these mutation sites are responsible for the stability of the N-terminal portion released. The crystal structure of inactive cathepsin D at pH showed that the N-terminal portion is relocated into the active-site cleft and is stabilized by an interaction to the catalytic site Lee et al. 1998 . From this crystal structure data we suspect the second possibility to be more likely. Disulfide Linkages Disulpfide links are known to connect distal regions of the polypeptide chain and therefore have often been