Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 97, Issue 17, Pages 9408-9412Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.150084897
Keywords
hydroxyalkenal; protein redesign; sequential design
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The evolution of proteins for novel functions involves point mutations and recombinations of domains or structural segments. Mimicking this process by rational design in vitro is still a major challenge. The present report demonstrates that the active site of the enzyme glutathione transferase (GST) Al-l can be tailored for high catalytic efficiency with alkenals. The result is a >3,000-fold change in substrate selectivity involving a noteworthy change in preferred catalyzed reaction from aromatic nucleophilic substitution to Micael addition. The hydrophobic substrate binding pocket of GST A1-1 is formed by three structural modules, which were redesigned sequentially with four point mutations and the exchange of a helical segment. The substitutions were made to mimic first-sphere interactions with a substrate in GSTA4-4, which naturally has high activity with alkenals, These substrates are toxic lipid peroxidation products of pathophysiological significance, and glutathione conjugation is a route of their inactivation. The final product of the sequential redesign of GST Al-l, mutant GIMFhelix. had a 300-fold increase in catalytic efficiency with nonenal and a >10 times decreased activity with 1-chloro-2,4-dinitrobenzene. In absolute values, GIMFhelix is more efficient than wild-type CST A4-4 with some alkenal substrates, with a k(cat)/K-m value of 1.5 +/- 0.1 10(6) M-1 s(-1) for nonenal. The pKa value of the active-site Tyr-9 of GIMFhelix is 7.3 +/- 0.1, approaching the unusually low value of GST A4-4. Thus, rational redesign of the active-site region of an enzyme may be sufficient for the generation of efficient catalysts with altered chemical mechanism and novel selectivity.
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