Mutant enzyme structural mutation

Both types of mutations have been made in T4 lysozyme. The chosen mutations were Gly 77-Ala, which caused an increase in Tm of 1 °C, and Ala 82-Pro, which increased Tm by 2 °C. The three-dimensional structures of these mutant enzymes were also determined the Ala 82-Pro mutant had a structure essentially identical to the wild type except for the side chain of residue 82 this strongly indicates that the effect on Tm of Ala 82-Pro is indeed due to entropy changes. Such effects are expected to be additive, so even though each mutation makes only a small contribution to increased stability, the combined effect of a number of such mutations should significantly increase a protein s stability. 

This approach is not restricted to bacterial or viral cells. Mammalian cells under highly proliferating conditions can be cultured at increasing exposure to a compound in attempts to create resistant mutants. Alternatively, one can sometimes use a structural biology approach to predict amino acid changes that would abrogate inhibitor affinity from study of enzyme-inhibitor complex crystal structures. If the recombinant mutant enzyme displays the diminished inhibitor potency expected, one can then devise ways of expressing the mutant enzyme in a cell type of interest and look to see if the cellular phenotype is likewise abolished by the mutation. 

One of the important consequences of studying catalysis by mutant enzymes in comparison with wild-type enzymes is the possibility of identifying residues involved in catalysis that are not apparent from crystal structure determinations. This has been usefully applied (Fersht et al., 1988) to the tyrosine activation step in tyrosine tRNA synthetase (47) and (49). The residues Lys-82, Arg-86, Lys-230 and Lys-233 were replaced by alanine. Each mutation was studied in turn, and comparison with the wild-type enzyme revealed that each mutant was substantially less effective in catalysing formation of tyrosyl adenylate. Kinetic studies showed that these residues interact with the transition state for formation of tyrosyl adenylate and pyrophosphate from tyrosine and ATP and have relatively minor effects on the binding of tyrosine and tyrosyl adenylate. However, the crystal structures of the tyrosine-enzyme complex (Brick and Blow, 1987) and tyrosyl adenylate complex (Rubin and Blow, 1981) show that the residues Lys-82 and Arg-86 are on one side of the substrate-binding site and Lys-230 and Lys-233 are on the opposite side. It would be concluded from the crystal structures that not all four residues could be simultaneously involved in the catalytic process. Movement of one pair of residues close to the substrate moves the other pair of residues away. It is therefore concluded from the kinetic effects observed for the mutants that, in the wild-type enzyme, formation of the transition state for the reaction involves a conformational change to a structure which differs from the enzyme structure in the complex with tyrosine or tyrosine adenylate. The induced fit to the transition-state structure must allow interaction with all four residues simultaneously. 

It is interesting that although the Val-143— His mutation leads to a bulky side chain at the base of the hydrophobic pocket, the mutant enzyme exhibits only a 10 -fold loss of CO2 hydrase activity relative to the wild-type enzyme (Fierke et ai, 1991). In this mutant the Val-I43- His side chain packs differently in the pocket relative to the side chains of the Val-143—>Phe and Val-143- Tyr mutants (Alexander et ai, 1991). It is likely that differences in side-chain packing, as well as differences involving active-site solvent structure, are responsible for differences in enzyme-substrate association behavior among the residue-143 mutants of carbonic anhydrase II. 

The mutant porcine pepsins, T77D, G78(S)S79, and T77D/G78(S)S79, were purified by the same method as wild-type pepsin, and the purities of the enzymes were judged by SDS-PAGE. The NH2-terminal sequences of the mutants were the same as that of wild-type enzyme. The secondary structures of recombinant wild-type and mutant pepsins were analyzed by CD spectrometry to determine whether localized or global changes of structures were induced by the mutations. The CD spectral data showed that the spectra of the mutants were essentially superimposable on that of the wild-type enzyme. These results suggest that no major conformational alterations occurred in the mutant enzymes. 

CD spectroscopy has also provided valuable insight into the chemical stability and chemical denaturation of proteins. A recent study by Rumfeldt etal. examines the guanidinium-chloride induced denaturation of mutant copper-zinc superoxide dismutases (SODs). These mutant forms of the Cu, Zn-SOD enzyme are associated with toxic protein aggregation responsible for the pathology of amyotrophic lateral sclerosis. In this study, CD spectroscopy was used in conjunction with tryptophan fluorescence, enzyme activity, and sedimentation experiments to study the mechanism by which the mutated enzyme undergoes chemical denaturation. The authors found that the mutations in the enzyme structure increased the susceptibihty of the enzyme to form partially unfolded destabilized monomers, rather than the stable metaUated monomer intermediate or native metallated dimer. 

A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 x 10 -fold increase in the catalytic efficiency for a nonnative substrate, valine. The crystal structure of the mutant enzyme indicated a remodeled active site and altered subunit interface caused by the cumulative effects of mutations. Most amazingly, only one of the mutations directly contacts the substrate, which underscores our limited understanding of enzyme substrate specificity. These mutations would be difficult, if not impossible, to be identified and introduced to the mutant enzyme by a rational design approach. 

E. coli have also been determined (3, 9, 24-26). Figure 1 depicts the polypeptide backbone of the yeast enzyme, indicating the position of the heme, and the proximal and distal histidine residues. The structure can be divided into N- and C-terminal domains, and the heme is in a cavity at the domain interface. The substrate access channel is also at the domain interface and is discussed in Section V. The secondary structure is dominated by a-helices with only a small amount of jS-sheet in the proximal domain. The refined structures of the recombinant wild-type enzymes are essentially identical to that of the yeast enzyme, but small differences are observed in the mutants around the mutated residues (3). 

---