Role of the -Loop in the Activity, Substrate Specificity, and Structure of Class A 
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- 作者：Soojay Banerjee ; Ursula Pieper ; Geeta Kapadia ; Lewis K. Pannell ; and Osnat Herzberg
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：March 10, 1998
- 年：1998
- 卷：37
- 期：10
- 页码：3286 - 3296
- 全文大小：275K
- 年卷期：v.37,no.10(March 10, 1998)
- ISSN：1520-4995
文摘
The structure of class A 
-lactamases contains an 
-loopassociated with the active site, whichcarries a key catalytic residue, Glu166. A 16-residue -loopdeletion mutant of -lactamase fromStaphylococcus aureus PC1, encompassing residues163-178, was produced in order to examine thefunctional and structural role of the loop. The crystal structurewas determined and refined at 2.3 Å, andthe kinetics of the mutant enzyme was characterized with a variety of-lactam antibiotics. In general,the wild-type -lactamase hydrolyzes penicillin compounds better thancephalosporins. In contrast, thedeletion of the -loop led to a variant enzyme that acts only oncephalosporins, including third generationcompounds. Kinetic measurements and electrospray mass spectrometryrevealed that the first and thirdgeneration cephalosporins form stable acyl-enzyme complexes, except forthe chromogenic cephalosporin,nitrocefin, which after acylating the enzyme undergoes hydrolysis at a1000-fold slower rate than thatwith wild-type -lactamase. Hydrolysis of the acyl-enzymeadducts is prevented because the deletion ofthe -loop eliminates the deacylation apparatus comprising Glu166 andits associated nucleophilic watersite. The crystal structure reveals that while the overall fold ofthe mutant enzyme is similar to that ofthe native -lactamase, local adjustments in the vicinity of themissing loop occurred. The altered -lactamspecificity is attributed to these structural changes. In thenative structure, the -loop restricts theconformation of a -strand at the edge of the active site depression.Removal of the loop provides the-strand with a new degree of conformational flexibility, such thatit is displaced inward toward theactive site space. Modeled Michaelis complexes withbenzylpenicillin and cephaloridine show that theperturbed conformation of the -strand is inconsistent withpenicillin binding because of steric clashesbetween the -lactam side chain substituent and the -strand.In contrast, no clashes occur uponcephalosporin binding. Recognition of third generationcephalosporins is possible because the bulky sidechain substituents of the -lactam ring typical of these compoundscan be accommodated in the spacefreed by the deletion of the -loop.
-lactamases contains an -loopassociated with the active site, whichcarries a key catalytic residue, Glu166. A 16-residue -loopdeletion mutant of -lactamase fromStaphylococcus aureus PC1, encompassing residues163-178, was produced in order to examine thefunctional and structural role of the loop. The crystal structurewas determined and refined at 2.3 Å, andthe kinetics of the mutant enzyme was characterized with a variety of-lactam antibiotics. In general,the wild-type -lactamase hydrolyzes penicillin compounds better thancephalosporins. In contrast, thedeletion of the -loop led to a variant enzyme that acts only oncephalosporins, including third generationcompounds. Kinetic measurements and electrospray mass spectrometryrevealed that the first and thirdgeneration cephalosporins form stable acyl-enzyme complexes, except forthe chromogenic cephalosporin,nitrocefin, which after acylating the enzyme undergoes hydrolysis at a1000-fold slower rate than thatwith wild-type -lactamase. Hydrolysis of the acyl-enzymeadducts is prevented because the deletion ofthe -loop eliminates the deacylation apparatus comprising Glu166 andits associated nucleophilic watersite. The crystal structure reveals that while the overall fold ofthe mutant enzyme is similar to that ofthe native -lactamase, local adjustments in the vicinity of themissing loop occurred. The altered -lactamspecificity is attributed to these structural changes. In thenative structure, the -loop restricts theconformation of a -strand at the edge of the active site depression.Removal of the loop provides the-strand with a new degree of conformational flexibility, such thatit is displaced inward toward theactive site space. Modeled Michaelis complexes withbenzylpenicillin and cephaloridine show that theperturbed conformation of the -strand is inconsistent withpenicillin binding because of steric clashesbetween the -lactam side chain substituent and the -strand.In contrast, no clashes occur uponcephalosporin binding. Recognition of third generationcephalosporins is possible because the bulky sidechain substituents of the -lactam ring typical of these compoundscan be accommodated in the spacefreed by the deletion of the -loop.