Substitution rates between amino acids

The suggested relationship between numbers ol differences and evolutionary lime is not wholly secure. It assumes uniformity in the tale of clleeiive amino acid substitution, but this rale mas he neither iindorm with time, nor uniform in different pails of the polv vpticle chain. Differences in the rate of effective substitution along Ihe polypeptide chain may be due not only 10 restrictions imposed by the required tertiary structure, hut also to differences in the rate at which various parts of tile l).NA or the gene mutate. The evolution of hemoglobin mav he contrasted w ith that of cytochrome e in which approximately 500 of the molecule appears io have remained invariant purine the lime yeast arid man have evolved. 

With the determination of amino acid sequences of hemoglobins and cytochrome C from many mammalian sources, sequence comparisons revealed that the number of amino acid divergences between different pair of mammals seemed to be roughly proportional to the time since they had diverged from one another, as inferred from the fossil record (Holsinger, 2004). As a possible explanation, Zuckerkandl and Pauling (1965) propounded a molecular clock hypothesis in which they proposed the presence of a constant rate of amino acid substitution over time. 

Site-directed mutagenesis has become an important and widespread technique for the elucidation of structure-function relationships in proteins. However, the repercussions of mutations on both protein structure and catalysis are often subtle and, particularly in the case of mechanisms that require multiple catalytic steps, not always easily interpretable. Classical comparison of catalytic rate parameters between mutant and native enzymes where an amino acid substitution results in a change in the the rate-limiting step of a reaction are not necessarily valid (109). Thus, direct detection of reaction intermediates is an important means for assessing the effect of mutations on the mechanism and for accurately determining the role of various protein residues in catalysis. 

Models of Substitution Rates Between Amino Acids... 

In the 1960, it was noticed that substitutions in some amino acid sequences seemed to occur at a roughly constant rate over time (Zuckerkandl and Pauling, 1965). This is the well known molecular clock hypothesis. For a particular protein such as cytochrome c or myoglobin, it was noticed that there was a linear relationship between divergences of pairs of sequences, as measured by numbers of amino acid differences, and divergences of the species, as measured by dates from the fossil record. There is still considerable debate as to how accurate the molecular clock may be and as to how it might vary systematically depending on the species, type of protein, and kinds of substitutions that are counted. 

In a collaboration between the Abelson and Hecht labs [56b], a series of noncoded amino acids were introduced into dihydrofolate reductase (DHFR) to probe substrate binding and the requirement of an aspartic acid residue for catalytic competence. When aspartic acid analogs mono- or disubstituted at the )0-carbon were substituted for the active site aspartic acid residue, the mutant DHFRs were still able to catalyze the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate at 74 - 86 % of the wild-type rate. While hydride transfer from NADPH is not the rate-limiting step for the wild-type enzyme at physiological pH, a kinetic isotope experiment with NADPD indicated that hydride transfer had likely become the rate-limiting step for the mutant containing the )0,)0-dimethylaspartic acid. 

The initiating reaction between aldoses and amines, or amino acids, appears to involve a reversible formation of an N-substituted aldosyl-amine (75) see Scheme 14. Without an acidic catalyst, hexoses form the aldosylamine condensation-product in 80-90% yield. An acidic catalyst raises the reaction rate and yet, too much acid rapidly promotes the formation of 1-amino-l-deoxy-2-ketoses. Amino acids act in an autocat-alytic manner, and the condensation proceeds even in the absence of additional acid. A considerable number of glycosylamines have been prepared by heating the saccharides and an amine in anhydrous ethanol in the presence of an acidic catalyst. N.m.r. spectroscopy has been used to show that primary amines condense with D-ribose to give D-ribopyrano-sylamines. ... 

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