RTEM /3-lactamase

D. G. Brenner, J. R. Knowles, Penicillanic Acid Sulfone Nature of Irreversible Inactivation of RTEM /3-Lactamase from Escherichia colV, Biochemistry 1984, 23, 5833-5839. 

Ribosome display (Figure 2) has been used to select protein-binding partners but, to our knowledge, never been used to find the unknown protein-binding partner for a small molecule.268 272-275 Fiowever, a recent paper by Pluckthun clearly indicates that this is possible. In this report, the authors show, for the first time, that it is possible to use ribosome display to select for catalytic activity based on catalytic turnover (directed evolution). In their experiment, they displayed variants of RTEM-/3-lactamase and used biotinylated ampicillin sulfone (80) to select for catalytically active variants (Figure 3)276... 

Charnas RL, Knowles JR. Inactivation of RTEM 3-lactamase from Escherichia coli by clavulanic acid and 9-deoxyclavulanic acid. Biochemistry 1981 20 3214-3219. 

Y. Song, R. Kluger, Benzylpenicillin Methyl Phosphate. A Penicillin Prodrug That Inactivates RTEM /L Lactamase , Bioorg. Med. Chem. Lett. 1994, 4, 1225- 1228. 

Sigal, I.S. Harwood, B.G. Arentzen, R. Thiol-beta-lactamase—replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue. Proc. Natl. Acad. Sci. USA. 1982, 79 (23), 7157-7160. 

Folding Equilibrium Studies. E. coli RTEM p-lactamase is a monomeric protein. Its amino acid sequence has been determined (79). It has one disulfide bond between the residues Cys S and Cys. The presence of four tyrosines and four tryptophans allows the use of spectroscopic method for the conformational characterization of the enzyme. In this study, the effect of denaturants on the unfolding of p-lactamase was determined from activity measurements, difference spectroscopy and fluorescence intensity measurements. 

In the case of penicillin G, the outward rotation about the C3-C4 bond can occur vithout conflict with protein side chains. It relaxes the strained conformation along this bond previously enforced by the four-membered ring. Moreover, this rotation improves hydrophobic contact of the dimethyl and the sulfur part of the thiazolidine ring of penicillin with two leucine side chains, thus offering an additional driving force for the outward rotation. Experimental evidence is provided by the observed rotation of 35.2° in this direction about the C3-C4 bond that occurs in the acyl-enzyme complex formed between benzyl penicillin and a mutant class A beta-lactamase (RTEM-1) blocked in deacylation [9]. 

Class C )3-Lactamase Citrobacter freundii 1982 Class A /3-Lactamase RTEM-3 ... 

Over 80 different (3-lactamases are now known. One classification is a system that divides the enzymes into three classes A, B, and C. Classes A and C are active-site serine enzymes. The serine residue in class A enzymes is at position 70. This class contains four major (3-lactamases 749/C (from B. licheniformis), PCI (from S. aureus), 569/H P-lactamase I (from B. cereus), and PBR322 and RTEM (from E. coli). As with other serine-type hydrolytic enzymes (acetylcholinesterase, trypsin), the mechanism of action requires initial formation of an acylated enzyme, in this case acylation of ser-70 followed by hydrolysis of the derivative to regenerate the enzyme ... 

Figure 20 Interfacial surface between trial docking of rtem (beta lactamase) and blip (beta lactamase inhibitory protein). Molecular surfaces are low order spherical harmonic. " Interfiacial surface is computed interactively on-the-fly while docking molecules are moved relative to each other. Color coding on molecular surfaces indicate shape properties. (Image courtesy of Arthur J. Olson, The Scripps Research Institute, La Jolla, CA)...

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