Organization of an Efficient Carbonic Anhydrase: Implications for the Mechanism Based on Structure-Function Studies of a T199P/C206S Mutant

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• 作者：Shenghua Huang ; Bjö ; rn Sjö ; blom ; A. Elisabeth Sauer-Eriksson ; and Bengt-Harald Jonsson
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：June 18, 2002
• 年：2002
• 卷：41
• 期：24
• 页码：7628 - 7635
• 全文大小：317K
• 年卷期：v.41,no.24(June 18, 2002)
• ISSN：1520-4995

文摘

Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)¹reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206Svariant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanateand mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All threeligands display novel binding interactions to the T199P/C206S mutant. The mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-mercaptoethanol moleculebinds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanatebinds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated bindingto the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at thepositions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area ismore hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part ofthe cavity normally occupied by the so-called deep water (Wat338). The observation of a new bindingsite for bicarbonate has implications for understanding the mechanism by which the main-chain aminogroup of Thr199 acquired an important role for orientation of the substrate during the evolution of theenzyme.