Mutation acid-base catalysis

Perrotta AT, Shih I, Been MD. Imidazole rescue of a cytosine mutation in a self-cleaving rihozyme. Science 1999 286 123-126. Nakano S, Chadalavada DM, Bevilacqua PC. General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme. Science 2000 287 1493-1497. 

A synchronous transfer of two protons, which in reaction (9.14) competes with a two-step process, is in some cases the predominant proton exchange mechanism. Such double proton migrations play an important role in many chemical and biochemical reactions in which the steric hindrances impeding proton transfer in a substrate molecule are removed thanks to the double proton exchange between substrate and enzyme [79]. The double proton transfers determine the mechanism of the bifunctional acid-base catalysis[80, 81]. The interest in the mechanism of double proton migrations in the H-bound complexes became especially keen after Lowdin [82] advanced in 1963 the hypothesis to the effect that it is precisely such processes in the DNA molecules that underlie the nature of spontaneous mutations. 

In most countries, about one third of the mutations that are identified wfll not have been reported previously in AIP and may represent rare polymorphisms rather than disease-specific mutations. Criteria that suggest that such novel mutations cause disease include production of a frameshift or stop codon, the absence of any other sequence abnormality in the gene, segregation with disease, and nonconservative change of an amino acid residue that is conserved between species and/or known to have a functional role in catalysis. Mutations of consensus bases in splice sites are also likely to be disease specific, but ideally all putative splicing defects should be confirmed by analysis of mRNA. Proof that a missense mutation causes disease may require expression and characterization of the mutant enzyme in a prokaryotic or eukaryotic vector. 

Interactions with carboxylates in the +1 site seem to modulate trans-glycosylation activity in the human pancreatic a-amylase as expected, nucleophile mutants were inactive, acid-base catalyst mutants showed reduced deglycosylation rates, but much reduced glycosylation rates only with oligosaccharide substrates, rather than maltotriosyl fluoride. However, mutation of the auxiliary Asp300 impaired transglycosylation, rather than catalysis. 

The reduction of the pyrimidine to dihydropyrimidine is the reverse of the oxidation reaction carried out by DHODs. The structure of the FMN/pyrimidine-binding site is very similar to the structure of L. lactis DHODs. Three Asn residues form hydrogen bonds with the nitrogens and carbonyls of the pyrimidine analogous to DHODs. DPD has an active site cysteine proposed to act in acid/base chemistry similar to Class 1 DHODs. When mutated to alanine, only 1% of the wild-type activity was retained, indicating the importance of this residue in catalysis. Secondary tritium isotope effects using 5- H-uracil were determined in both H2O and D2O an inverse isotope effect was observed in H2O and the value became more inverse in D20. " This was taken as evidence of a stepwise mechanism in which hydride transfer to C6 is followed by protonation at C5. 

First assumptions as to the possibility of hydrogen changing its position in the process of transformation of one chemical particle into another one were voiced nearly 200 years ago [31], and the mechanism of this enormously important reaction has been attracting attention ever since. The reason for this interest is obvious seeing that the proton transfer reactions underlie the acid-base equilibria and determine the mechanisms of a broad range of biological phenomena such as the transport across membranes, enzymic catalysis, photosynthesis, the formation of the ATP acid, spontaneous mutations etc. 

The catalytic center is formed by residues from both lobes. Sequence comparisons, mutation experiments and biochemical studies indicate an essential fimction in catalysis of phosphate transfer for the conserved amino acids Lys72, Aspl66 and Aspl84 (numbering of PKA). However, the catalytic mechanism of phosphate transfer is not definitely established. It is generally assumed that Aspl66, which is invariant in all protein kinases, serves as a catalytic base for activation of the Ser/Thr hydroxyl and that the reaction takes place by an in-line attack of the Ser-OH at the y-phosphate. 

Wang et al. (1994) analyzed by MD the roles of the "double catalytic triad" in papain catalysis, based on the structure of the enzyme, which is not completely known from crystallography (Kamphuis et al., 1984) due to the oxidation state of Cys-25 (present as cysteic acid in the crystal). Stochastic boundary MD (Brooks and Karplus, 1983) was carried out on the whole enzyme + 350 water molecules. Three "layers" were treated according to their distance from the sulfur atom of Cys-25 - atoms within 12A, atoms between 12-16A and the more distant atoms were kept fixed. CHARMM forcefield was employed. The active site geometry was examined as a function of pH, for various mutual states of S-/SH and Im/ImH+. In addition, the mutations of Asp-158 (Menard et al., 1991) were studied. 

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