Lysozyme enzyme, mutagenesis

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... 

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. 

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... 

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