Mutant enzymes structure determination

Figure 1 In a typical example of the perturbation approach, the strength of two selected Tyr-Glu hydrogen bonds in the structure of SNase was systematically studied by creating mutant enzymes and determining their folding energies.

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. 

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. 

Crystal structure determinations of MnSODs from organisms ranging from E. coli to humans have been reported. Structural determinations of note include those by Jameson et al. on the E. coli enzyme and mutant forms of this enzyme with atomic resolution,a cambialistic superoxide dismutase from Porphyromonas gingivalis, and mutant forms of the human enzyme the Q143N, and Q143A mutants.The coordination sphere of the... 

X-ray crystal structures of glutamine synthetase from both Salmonella typhimuriuni and Mycobacterium tuberculosis are very similar. Structures of wild type enzymes and of active site mutants have been determined. All structures have been solved with Mn in the active site. There are twelve identical subunits arranged in two face-to-face symmetrical hexamers. The active sites are in funnel-shaped open-ended cavities located between adjacent subunits of the hexamer. These cavities are 45 A long, 30 A wide at the outer end, and 10 A wide at the inner end and the active site with the two Mn " ions is approximately halfway down the cavity. The metal-metal distance is 5.8 A. The more tightly bound Mn is coordinated to the side chains of Glu-131, Glu-212, Glu-220, and two water molecules, one of which is shared by both metal ions. Glu-129, Glu-357, His-269, and two additional water molecules are bound to the Mn + at the lower affinity site. A schematic view of the active site metal coordination is shown in Figure 36. 

In concert, structure determinations and enzymological studies for catalytic rates and product distributions with structurally varied aldehydes of native enzymes and numerous active-site mutants have allowed us to derive a conclusive blueprint for the catalytic cycle of FucA (Fig. 2.2.5.2). The proposed mechanism, which has general implications for other metal-dependent aldolases, is able to rationalize all key stereochemical issues successfully ]15]. Independent work by other groups has recently provided further insight into related proteins with Fru A and TagA specificity [16]. 

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. 

Hydrophobicity indices are fromKyte, J. Doolittle, R. F. A,J.Mol. Biol., 157,105-132(1982). dNI) means that the values are not determined, because the mutant enzymes from denatured structure. CNA means that the mutant enzymes were not analyzed, because they were not secreted in the culture medium. 

In order to determine the relationship between protein structure and function and to create mutant enzymes with altered properties useful for biotechnology and cancer therapy, a directed evolution approach has been explored and novel proteins developed for Pol I DNA polymerase enzymes thymidylate synthase, thymidine kinase and 06-alkylguanine-DNA alkyltransferase. In every case the creation of a large variety of altered proteins has been achieved, and the emerging picture is that even highly conserved proteins can tolerate wide-spread amino acid changes at the active site with-... 

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