Structural Aspects of Mutant Enzymes

Thermodynamic stability is a global property of the enzyme structure, and contributions of individual amino acids toward the free energy of folding are additive and highly cooperative. Analysis of mutant proteins has defined the contributions of various amino acids toward the overall stability of the protein. Replacements which alter the formation of ion pairs, hydrogen bonds, van der Waals contacts, or hydrophobic interactions each tend to destabilize the folded protein by a qualitatively comparable amount (14). In one approach to this problem, site-specific substitution of amino acids has provided new approaches toward dissecting the kinetic mechanisms of protein folding (27). 

New insights into protein folding have been obtained by analysis of the locations of temperature-sensitive mutations in T4 lysozyme (70). These particularly destabilizing mutations appear to have only one feature in common they occur at sites of low mobility and low solvent accessibility in the folded state, as illustrated in Fig. 1. Any substitution of an amino acid at these locations perturbs the 

The stability gained by the added disulfide bond has been lower than might be anticipated and somewhat variable. Disulfide linkages in wild-type proteins have special geometries which may be difficult to achieve in an engineered protein the modest reduction in entropy in forming the disulfide may be offset by unfavorable distortion in the protein structure [see Ref, (14) and references cited 

It has also been possible to modulate enzyme function (29) by introduction of a disulfide bridge spanning the active site cleft of T4 lysozyme (Fig. 1). In order to avoid possible thiol-disulfide interchange with Cys-S4 and Cys-97 in the native structure, these two residues were converted to threonine and alanine, respectively, with no loss in the activity or stability of the enzyme. The latter protein was then further modified by replacing Thr-21 and Thr-42 by cysteines that spontaneously oxidized to the desired disulfide. This oxidized enzyme form exhibited no detectable activity, although some activity (7% of wild type) was restored on reduction of the linkage. This represents a novel use of the disulfide bond to modulate catalytic activity. 

Reduction in the area of exposed hydrophobic surfaces can also enhance thermodynamic stability. Chothia has estimated a proportionality constant of 24 cal/ mol of hydrophobic free energy per square angstrom of solvent-exposed surface area (32). Substitutions at Ile-3 of T4 lysozyme enhance the stability by amounts that agree surprisingly well with this prediction (33). However, there is some debate over the choice of the proper hydrophobicity scale to quantitate the contributions of each hydrophobic residue, and it is perhaps an oversimplification to expect such a simple relationship to hold for all amino acids (34). 

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