Site-directed mutagenesis lysozyme

Lavoie, T. B., Drohan, W. N. and Smith-Gill, S. J. (1992), Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme , f. Immunol., 148, 503-513. 

A pathway (Scheme I) (8.9) for the hydrolysis of oligoglycosides by lysozyme that differs from the previously accepted mechanism (Scheme II) (3,10-12) is described in this section. The alternative pathway, suggested by results of a 55-ps MD simulation of the lysozyme (GlcNAc)e complex (1), is consistent with the available experimental data and with stereoelectronic considerations. Experimental data have demonstrated that Glu 35 and Asp 52 are essential, as shown by recent site-directed mutagenesis results (13.) which corroborate chemical modification studies (3.14 and references cited therein), and that the reaction proceeds with retention of configuration at Ci Q and references cited therein). A fundamental feature of the alternative pathway is that an endocyclic bond is broken in the initial step, in contrast to the exocyclic bond cleavage in the accepted mechanism. 

This formalism may also be valuable in addressing protein unfolding, DNA unwinding, cold denaturation, and effects of site-direct mutagenesis on lysozyme unfolding. ... 

The distance measured by EPR is the point dipole distance between the two paramagnetic centres. When applied to structures of spin-labelled biomolecules the desired distance is between sites on the biomolecules. A key question is the conformation and conformational flexibility of the spin label. X-ray crystal structures of four spin-labelled derivatives of T4 lysozyme have been reported.54 Preferred rotational conformations of the linkage between the cysteine introduced by site-directed mutagenesis and the spin label were observed. The electron density associated with the nitroxide ring in different mutants was inversely correlated with the mobility of the label observed in fluid solution EPR spectra. 

The preferred modem procedure for investigating an enzyme mechanism is to identify a protein in a genome sequence, clone and express it, solve the crystal and/or solution structures, guess the mechanism, and then confirm it by site-directed mutagenesis and kinetics. The mechanism of lysozyme almost illustrates that procedure. Although the classical protein chemistry on lysozyme predated the molecular genetics, the kinetic studies were stimulated by the x-ray crystallography, and the previously unknown mechanism almost leapt from the... 

Since there are strict stereochemical requirements for the relative positions and orientations of the two participating cysteine residues,11 addition of new disulfides to existing proteins by site-directed mutagenesis has not always produced the desired increase in stability. Introduction of disulfide bonds has been attempted for phage T4 lysozyme,4-71 phage A repressor,81 dihydrofolate reductase,91 and subtilisins.10-131 Among them the most extensive study has been performed on T4 lysozyme, and enhancement of protein stability has been successful. 

Some studies have been reported where individual aromatic groups of a protein have been systematically replaced using site-directed mutagenesis, allowing the contributions of the individual chomophores to be determined. Craig et al. [106] have systematically relaced all the Trp moieties in interleukin-ip (IL-lp). Earlier work of Elwell and Schellman [107] replaced the tryptophan in T4 lysozyme with tyrosine. 

In summary, we have shown that site-directed mutagenesis is an important tool which allows precise investigation into the detailed chemistry of protein-protein interaction. HEL has proved to be ideal for these studies. Monoclonal antibodies, X-ray crystal structures of protein complexes, and evolutionary lysozyme variants found in nature provide an excellent system for understanding the molecular basis of protein recognition. Site-directed mutagenesis offers the ability to alter a protein at a specific site. Interpretation of the results within the context of an X-ray crystal structure allows the quantitative assignment of the free energy contributions from individual amino adds to the stability of the complex. 

With tree analysis it is possible to infer sequences of ancestral proteins and probable evolutionary pathways to their modern descendants.1 The advent of site-directed mutagenesis makes possible the recreation of evolutionary intermediates based on these predictions. One may then compare the properties of reconstructed intermediates with one another and with proteins from contemporary creatures. These comparisons provide a way of testing theories about the mechanism of molecular evolution. For example, this approach has provided a new criterion for distinguishing between neutral and nonneutral events.2 This chapter describes the use of site-directed mutagenesis to recreate ancestral lysozymes and presents methods of evaluating their properties. 

The CL variants are constructed by site-directed mutagenesis of the cDNA for chicken egg white lysozyme and heterologously expressed in yeast behind a regulated hybrid promoter.6... 

In contrast, in phage T4 lysozyme a thermolabile mutant was found in which one single amino acid was exchanged Thr- Ile 157, and the thermal melting point dropped from 42°C for the wild-type enzyme to 31 °C for the mutant. Systematic exchange of the amino acid in position 157 by site-directed mutagenesis led to the... 

Genetic engineering of hen egg-white lysozyme has been used by Kirsch el al. (1989) as an approach to studying the structure—function relationships of lysozyme. Thus, they offer evidence from site-directed mutagenesis of cloned lysozyme (expressed in yeast), that Asp-52 and Glu-35 are vital for the expression of lysozyme. However, it is curious that conversion of Asp-52 to the amide resulted in a form of the enzyme that still had 5% of the normal activity. Conversion of Glu-35 to the amide, on the other hand, resulted in a lysozyme that was devoid of all activity. It was demonstrated by mutagenesis of Asp-IOI to Gly that the ionization of this residue contributes thermodynamically to the association of lysozyme with the inhibitor chitotriose. 

Blake CCF, lohnson LN, Mair GA, North ACT, Philhps DC, Sharma VR. Lysozyme. Proc. Roy. Soc. Ser. B 1967 167 378-381. Lesburg CA, Zhai G, Cane DE, Christianson DW. Crystal structure of pentalenene synthase mechanistic insights on terpenoid cychzation reactions in biology. Science 1997 277 1820-1824. Seemann M, Zhai G, de Kraker IW, Paschall CM, Christianson DW, Cane DE. Pentalenene synthase. Analysis of active site residues by site-directed mutagenesis. 1. Am. Chem. Soc. 2002 124 7681-7689. 

Application of site-directed mutagenesis to establish significance of individual amino acids in catalysis has become a common tool in enzymology, although relatively rare with glycoside hydrolases and glycosyltransferases, which are limited to date to studies on /3-galactosidase (136-140), lysozyme (141), and invertase (142). 

Before the introduction of site-directed mutagenesis, chemical modification was the predominant means of altering a specific amino acid the earliest report with lysozyme was from Parsons and Raftery (241), who prepared the ethyl ester derivative of Asp-52 by reaction with triethyloxonium fluoroborate the modified enzyme lost catalytic function but not substrate affinity. An ethyleneimine reaction product of Asp-52 has also been prepared with similar effects on catalysis (242). Sharon and co-workers modified and then regenerated Asp-52 to eliminate concern that inactivation results from experimental manipulation rather than specific amino acid modification (243, 244). Thus, Asp-52 was first esteri-fied with an epoxypropyl-jS-glycoside derivative of di-(/V-acetyl-D-glucosamine), then reduced to homoserine or hydrolyzed to return the free aspartate. Both the... 

TABLE 11.10 Studies on catalytic residues of lysozymes by site-directed mutagenesis... 

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