Improving the Catalytic Activity of a Thermophilic Enzyme at Low Temperatures
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- 作者：Astrid Merz ; Muh-ching Yee ; Halina Szadkowski ; Gü ; nter Pappenberger ; Andreas Crameri ; Willem P. C. Stemmer ; Charles Yanofsky ; and Kasper Kirschner
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：February 8, 2000
- 年：2000
- 卷：39
- 期：5
- 页码：880 - 889
- 全文大小：345K
- 年卷期：v.39,no.5(February 8, 2000)
- ISSN：1520-4995
文摘
Enzymes from thermophilic organisms often are barely active at low temperatures. To obtaina better understanding of this sluggishness, we used DNA shuffling to mutagenize the trpC gene, whichencodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutantsproducing more active protein variants were selected by genetic complementation of an Escherichia colimutant bearing a trpC deletion. Single amino acid changes and combinations of these changes improvedgrowth appreciably. Five singly and doubly altered protein variants with changes at the N- and C-termini,or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymaticcatalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purifiedvariant proteins correlated with the corresponding growth rates, showing that the turnover number wasthe selected trait. Although the affinities for both the substrate and the product decreased appreciably inmost protein variants, these defects were offset by the accumulation of high levels of the enzyme's substrate.Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme'sturnover number at low temperatures is limited by the dissociation of the enzyme-product complex. Incontrast, representative protein variants bind and release the product far more rapidly, shifting the bottleneckto the preceding chemical step. The turnover number of the parental enzyme increases with temperature,suggesting that its structural rigidity is responsible for its poor catalytic activity at low temperatures. Insupport of this interpretation, the rate of trypsinolysis or of thermal denaturation is accelerated significantlyin the activated protein variants.