Enzymes mutant

As these experiments with engineered mutants of trypsin prove, we still have far too little knowledge of the functional effects of single point mutations to be able to make accurate and comprehensive predictions of the properties of a point-mutant enzyme, even in the case of such well-characterized enzymes as the serine proteinases. Predictions of the properties of mutations using computer modeling are not infallible. Once produced, the mutant enzymes often exhibit properties that are entirely surprising, but they may be correspondingly informative. 

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. 

It may be necessary and possible to achieve a good Brf nsted relationship by adding another term to the equation, as Toney and Kirsch did in correlating the effects of various amines on the catalytic activity of a mutant enzyme. A simple Brf nsted plot failed, but a multiple linear regression on the variables pKa and molecular volume (of the amines) was successful. 

Garrett, R. M., et al., 1998. Human snlfite oxidase R160Q Identification of the mutation in a snlfite oxidase-deficient patient and expression and characterization of the mutant enzyme. Proceedings of the National Academy of Sciences HSA 95 6394—6398. 

Bacteria producing mutant enzymes grown in nutrient broth... 

Parallel to the modification of the catalytic performance in Baeyer-Villiger oxidations, random mutagenesis was successfully applied to improve the stereoselectivity of CHMOAcineto hi cascs of essentially racemic sulfoxide formation. In addition, enantiodivergent clones with >98% ee for both antipodal products were identified (Table 9.5) [205]. However, improvement in stereoselectivity of mutant enzymes was often accompanied by increased formation of sulfone. This effect can also be utilized to resolve racemic sulfoxides. 

The for the substrate UTP has been measured and does not show significant differences between wt and mutant enzymes. The model shows that the space available to substituents in positions 4 and 5 of the thiophene is limited, in agreement with SAR studies. Interaction with a number of basic and lipophilic residues bound... 

The CD spectrum of the C188S mutant is essentially the same as that of the wild-type enzyme, which reflects that the tertiary structure of this mutant changed little compared to that of the wild-type enzyme. Calculation of the content of secondary structure of the mutant enzyme based on J-600S Secondary Structure Estimation system (JASCO) also showed that there is no significant change in the secondary structure of the mutant. The fact that the k value of this mutant is extremely small despite little change in conformation clearly indicates that Cysl88 is located in the active site. 

As expected, the Gly74Cys single mutant enzyme exhibited racemase activity to some arylpropionates as summarized in Table 4. In general, good substrates... 

M28. Miwa, S and Fujii, H., Molecular basis of erythroenzymopathies associated with hereditary hemolytic anemia Tabulation of mutant enzymes. Am. J. Hematol. 51,122-132 (1996). 

Byrnes VW, Sardana W, Schleif WA, Condra JH, Waterbury JA, Wolfgang JA, Long WJ, Schneider CL, Schlabach AJ, Wolanski BS, Graham DJ, Gotlib L, Rhodes A, Titus DL, Roth E, Blahy OM, Quintero JC, Staszewski S, Emini EA. Comprehensive mutant enzyme and viral variant assessment of human immunodeficiency virus type 1 reverse transcriptase resistance to nonnucleoside inhibitors. Antimicrob Agents Chemother 1993 37 1576-1579. 

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. 

For scaled-up synthesis of larger amounts of metabolite, the following example is illustrative of the use of the BM3 mutant enzymes. 

The behaviour of the mutant enzymes where, for example, histidine-152 has been changed to alanine is compared with that of wild type enzymes.60 The 31P NMR chemical shift values and signal width for H152A mutant enzyme have shown the presence of two conformers open and closed forms of the enzyme that interconvert slowly on the NMR time scale. The tightness of the binding of the cofactor to the protein surface and its protonation state have been also discussed for intermediate Schiff bases in different steps of the catalytic cycle (Table 1). 

Xu, J. Voth, G. A., Free energy profiles for H+ conduction in the D-pathway of Cytochrome c oxidase a study of the wild type and N98D mutant enzymes, Biochim. Bio-phys. Acta 2006,1757, 852-859. 

In this example, structure-based design of inhibitors to the mutant enzyme may be undertaken using available X-ray structures and homology models. 

Wild-type and mutant enzymes Main a-linkage in glucan products Amino acid sequence around transition-state stabilizer D627 in GTFR... 

---