Structural and biochemical analysis of octopus S-crystallin

Autor: Yu-Tung Liu, 劉禹彤
Rok vydání: 2013
Druh dokumentu: 學位論文 ; thesis
Popis: 101
The major lens protein in cephalopods, S-crystallin, has been shown to have high amino acid sequence identity and structural similarity with glutathione S-transferase (GST). GST acts as a detoxification enzyme in biological system, which catalyzes the conjugation of reduced glutathione (GSH) with various endogenous and xenobiotic compounds. Differently, S-crystallin only shows very little GST activity. To find out the residues which incorporate the variations, we have identified several multiple mutations which base on several important single mutations in our current studies (D101A, D101N, L100F, M104V, and Q108F). By the way, different to GST, S-crystallin has a 16-residue long loop between the helix 4 and 5. Previous studies suggested that it may interfere the entering of substrate. We also try to truncate the loop .To test its influence. In our studies, the mutation of Gln108 to Phe of S-crystallin had a 40-fold increase in catalytic activity (higher kcat), compared with wild-type protein, while the mutation also resulted in lower binding affinity for GSH (higher Km, GSH). On the other hand, L100F, D101N and M104V mutants increased the binding affinity for CDNB. Our recent studies have found that D101A/Q108F double mutation shows higher kcat, and L100F/D101N/M104V/Q108F quadruple mutation lead to lower Km, CDNB. Surprisingly, the results of loop truncation suggested that the existence of the long loop can increase the binding of GSH to S-crystallin. Furthermore, wild- type S-crystallin shows substrate (GSH) protection in thermo-stability assay. Our studies suggested that the helix 4-5 loop insertion is a “permissive mutation” to gain GSH protection, while the four residues, Leu100, Asp101, Met104 and Gln108, play an important role in GSH protection, under the existence of the loop. It showed that S-crystallins, as the major lens proteins, are different from GST not only by reducing its GST activity, but also enhancing its structure stability by binding to GSH more tightly.
Databáze: Networked Digital Library of Theses & Dissertations
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