Enzyme:Substrate Hydrogen Bond Shortening during the Acylation Phase of Serine Protease Catalysis
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- 作者：Krisztiá ; n Fodor ; Veronika Harmat ; Richard Neutze ; Lá ; szló ; Szilá ; gyi ; Lá ; szló ; Grá ; f ; and Gergely Katona
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：February 21, 2006
- 年：2006
- 卷：45
- 期：7
- 页码：2114 - 2121
- 全文大小：352K
- 年卷期：v.45,no.7(February 21, 2006)
- ISSN：1520-4995
文摘
Atomic resolution (
1.2 Å) serine protease intermediate structures revealed that the strengthof the hydrogen bonds between the enzyme and the substrate changed during catalysis. The well-conservedhydrogen bonds of antiparallel 
-sheet between the enzyme and the substrate become significantly shorterin the transition from a Michaelis complex analogue (Pontastacus leptodactylus (narrow-fingered crayfish)trypsin (CFT) in complex with Schistocerca gregaria (desert locust) trypsin inhibitor (SGTI) at 1.2 Åresolution) to an acyl-enzyme intermediate (N-acetyl-Asn-Pro-Ile acyl-enzyme intermediate of porcinepancreatic elastase at 0.95 Å resolution) presumably synchronously with the nucleophilic attack on thecarbonyl carbon atom of the scissile peptide bond. This is interpreted as an active mechanism that utilizesthe energy released from the stronger hydrogen bonds to overcome the energetic barrier of the nucleophilicattack by the hydroxyl group of the catalytic serine. In the CFT:SGTI complex this hydrogen bondshortening may be hindered by the 27I-32I disulfide bridge and Asn-15I of SGTI. The position of thecatalytic histidine changes slightly as it adapts to the different nucleophilic attacker during the transitionfrom the Michaelis complex to the acyl-enzyme state, and simultaneously its interaction with Asp-102and Ser-214 becomes stronger. The oxyanion hole hydrogen bonds provide additional stabilization foracyl-ester bond in the acyl-enzyme than for scissile peptide bond of the Michaelis complex. Significantdeviation from planarity is not observed in the reactive bonds of either the Michaelis complex or theacyl-enzyme. In the Michaelis complex the electron distribution of the carbonyl bond is distorted towardthe oxygen atom compared to other peptide bonds in the structure, which indicates the polarization effectof the oxyanion hole.
1.2 Å) serine protease intermediate structures revealed that the strengthof the hydrogen bonds between the enzyme and the substrate changed during catalysis. The well-conservedhydrogen bonds of antiparallel -sheet between the enzyme and the substrate become significantly shorterin the transition from a Michaelis complex analogue (Pontastacus leptodactylus (narrow-fingered crayfish)trypsin (CFT) in complex with Schistocerca gregaria (desert locust) trypsin inhibitor (SGTI) at 1.2 Åresolution) to an acyl-enzyme intermediate (N-acetyl-Asn-Pro-Ile acyl-enzyme intermediate of porcinepancreatic elastase at 0.95 Å resolution) presumably synchronously with the nucleophilic attack on thecarbonyl carbon atom of the scissile peptide bond. This is interpreted as an active mechanism that utilizesthe energy released from the stronger hydrogen bonds to overcome the energetic barrier of the nucleophilicattack by the hydroxyl group of the catalytic serine. In the CFT:SGTI complex this hydrogen bondshortening may be hindered by the 27I-32I disulfide bridge and Asn-15I of SGTI. The position of thecatalytic histidine changes slightly as it adapts to the different nucleophilic attacker during the transitionfrom the Michaelis complex to the acyl-enzyme state, and simultaneously its interaction with Asp-102and Ser-214 becomes stronger. The oxyanion hole hydrogen bonds provide additional stabilization foracyl-ester bond in the acyl-enzyme than for scissile peptide bond of the Michaelis complex. Significantdeviation from planarity is not observed in the reactive bonds of either the Michaelis complex or theacyl-enzyme. In the Michaelis complex the electron distribution of the carbonyl bond is distorted towardthe oxygen atom compared to other peptide bonds in the structure, which indicates the polarization effectof the oxyanion hole.