Reduction enzyme mutation

Mutations in another region, the second cytoplasmic loop between M2 and M3 in Ca-ATPase of sarcoplasmic reticulum (Thr ->Ala, Gly -t Ala, and Glu Gln) also result in a complete loss of Ca-transport and Ca-ATPase activity associated with a dramatic reduction in the rate of phosphoenzyme turnover [96]. These mutations do not affect the affinity of the enzyme for Pj and therefore resemble the Pro mutants [123] in that they affect only the E1P-E2P conformational change and not the affinities for ATP, Ca or Pj. 

Homocystinuria can be treated in some cases by the administration of pyridoxine (vitamin Bs), which is a cofactor for the cystathionine synthase reaction. Some patients respond to the administration of pharmacological doses of pyridoxine (25-100 mg daily) with a reduction of plasma homocysteine and methionine. Pyridoxine responsiveness appears to be hereditary, with sibs tending to show a concordant pattern and a milder clinical syndrome. Pyridoxine sensitivity can be documented by enzyme assay in skin fibroblasts. The precise biochemical mechanism of the pyridoxine effect is not well understood but it may not reflect a mutation resulting in diminished affinity of the enzyme for cofactor, because even high concentrations of pyridoxal phosphate do not restore mutant enzyme activity to a control level. 

Point mutations can occur when one base is substituted for another (base substitution). Substitution of another purine for a purine base or of another pyrimidine for pyrimidine is called a transition, while substitutions of purine for pyrimidine or pyrimidine for purine are called transversions. Both types of base substitution have been identified within mutated genes. These changes lead to a codon change which can cause the wrong amino acid to be inserted into the relevant polypeptide and are known as mis-sense mutations. Such polypeptides may have dramatically altered properties if the new amino acid is close to the active center of an enzyme or affects the three-dimensional makeup of an enzyme or a structural protein. These changes, in turn, can lead to change or reduction in function, which can be detected as a change in phenotype of the affected cells. 

Bahnson et al. extended the series of mutations to include ones in which reductions occurred in the second-order rate constant / cat/ M by as much as a factor of 100. No substantial changes were observed in the primary isotope effects or their Swain-Schaad exponent. However, the precisely measured secondary isotope effects changed systematically as the rate constant decreased, such that the Swain-Schaad exponent decreased monotonically with decreasing fecat/ M from exponent of 8.5 for the L57F mutant (reactivity equivalent to the wild-type enzyme) to an exponent of 3.3 for the V203G mutant, slower by 100-fold. 

In some cases, mutation can lead to enhanced catalytic ability of the enzyme. Results for the mutation Thr-51 to Pro-51 (Wilkinson et al 1984) have been mentioned previously. The results for this and for the mutation Thr-51 to Ala-51 (Fersht e/ al., 1985) are also shown in Table 18. These mutations and that of Thr-51 to Cys-51 have been studied in some detail (Ho and Fersht, 1986). In each case it is found that the transition state is stabilized for formation of tyrosine adenylate from tyrosine and ATP within the enzyme the mutant Thr-51 to Pro-51 increases the rate coefficient for the reaction by a factor of 20. However, the enzyme-bound tyrosine adenylate is also stabilized by the mutation and this results in a reduced rate of reaction of tyrosine adenylate with tRNA (48), the second step in the process catalysed by tyrosine tRNA synthetase. Overall, therefore, the mutants are poorer catalysts for the formation of aminoacyl tRNA. The enzyme from E. coli has the residue Pro-51 whereas Thr-51 is present in the enzyme from B. stearothermophilus. The enzyme from E. coli is more active than the latter enzyme in both the formation of tyrosine adenylate and in the aminoacyla-tion of tRNA (Jones et al., 1986b). It is therefore suggested (Ho and Fersht, 1986) that the enzyme from E. coli with Pro-51 must additionally have evolved ways of stabilizing the transition state for formation of tyrosine adenylate without the concomitant stabilization of tyrosine adenylate and reduction in the rate of aminoacylation of tRNA found for the Pro-51 mutant. 

We have used a series of biocatalysts produced by site-directed mutations at the active site of L-phenylalanine dehydrogenase (PheDH) of Bacillus sphaericus, which expand the substrate specificity range beyond that of the wild-type enzyme, to catalyse oxidoreduc-tions involving various non-natural L-amino acids. These may be produced by enantiose-lective enzyme-catalysed reductive amination of the corresponding 2-oxoacid. Since the reaction is reversible, these biocatalysts may also be used to effect a kinetic resolution of a D,L racemic mixture. ... 

This electrode is unique in that the bilirubin oxidase is active at neutral pH, whereas the laccase cited above is not, even though the redox potential of laccase is somewhat higher. Additionally, the bilirubin oxidase is much less sensitive to high concentrations of other anions such as chloride and bromide, which deactivate laccase. It was shown that mutations of the coordination sphere of bilirubin oxidase led to an increased redox potential of the enzyme, which increased current density and reduced current decay to 5%/day over 6 days at 300 rpm. The latter improvement was attributed to improved electrostatic attraction between the enzyme and the redox polymer. An electrode made with high-purity bilirubin oxidase and this redox polymer has recently been shown to outperform a planar platinum electrode in terms of activation potential and current density of oxygen reduction. ... 

Inactivating LPL mutations lead to a > 60% reduction in LPL activity in postheparin plasma from homozygous individuals [49]. Mutations that result in only reduced LPL enzyme activity usually show less reduction in the activity assay, even in homozygous patients [64]. Heterozygous carriers of a mutation have no reduction in LPL activity and show no clinical signs. 

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