Mutant, specificity pocket

Model building also predicts that the Ala 216 mutant would displace a water molecule at the bottom of the specificity pocket that in the wild type enzyme binds to the NH3 group of the substrate Lys side chain (Figure 11.12). The extra CH3 group of this mutant is not expected to disturb the binding of the Arg side chain. One would therefore expect that the Km for Lys... 

Asp 189 at the bottom of the substrate specificity pocket interacts with Lys and Arg side chains of the substrate, and this is the basis for the preferred cleavage sites of trypsin (see Figures 11.11 and 11.12). It is almost trivial to infer, from these observations, that a replacement of Asp 189 with Lys would produce a mutant that would prefer to cleave substrates adjacent to negatively charged residues, especially Asp. On a computer display, similar Asp-Lys interactions between enzyme and substrate can be modeled within the substrate specificity pocket but reversed compared with the wild-type enzyme. 

Mutations in the specificity pocket of trypsin, designed to change the substrate preference of the enzyme, also have drastic effects on the catalytic rate. These mutants demonstrate that the substrate specificity of an enzyme and its catalytic rate enhancement are tightly linked to each other because both are affected by the difference in binding strength between the transition state of the substrate and its normal state. 

Adding a charge. In chymotrypsin, a mutant was constructed with Ser 189, which is in the bottom of the substrate specificity pocket, changed to Asp. What effect would you predict for this Ser 189 Asp 189 mutation ... 

T. Clackson, D.A. Holt, Investigating protein-ligand interactions with a mutant FKBP possessing a designed specificity pocket, J. Med. Chem. 2000, 43,1135-1142. 

The selectivity of the interaction of Nef with the Hck-SH3 domain is based on the interaction that the RT loop of the SH3 domain can form by extending over the surface of Nef. The RT loop is a remarkably variable and flexible structure, which in Src kinases can be differentially stabilized by networks of hydrogen-bonding interactions, according to its particular sequence (121). These differences may at least in part account for the selectivity of the Nef-SH3 interaction by stabilizing a particular conformation of the loop necessary for an optimal interaction with Nef. The character of this interaction is hydrophobic the side chain of Ile-96 of the Fyn-SH3 R—>I mutant inserts into an exposed, remarkably hydro-phobic crevice between the aA and aB helices of Nef. A correlation exists between the character of residues contributing to this specificity pocket and the ability of HIV-1, HIV-2, or SIV to interact with different Src family SH3 domains (124). The importance of this interaction is confirmed by the selection of RT-loop mutants of the Hck-SH3 domain that bind to Nef with affinities up to 40-fold higher than those of parental... 

The results of kinetic and X-ray crystallographic experiments on mutant carbonic anhydrases II, in which side-chain alterations have been made at the residue comprising the base of the hydrophobic pocket (Val-143), illuminate the role of this pocket in enzyme-substrate association. Site-specific mutants in which smaller hydrophobic amino acids such as glycine, or slightly larger hydrophobic residues such as leucine or isoleucine, are substituted for Val-143 do not exhibit an appreciable change in CO2 hydrase activity relative to the wild-type enzyme however, a substitution to the bulky aromatic side chain of phenylalanine diminishes activity by a factor of about 10 , and a substitution to tyrosine results in a protein which displays activity diminished by a factor of about 10 (Fierke et o/., 1991). 

G. DeSantis, X. Shang, and J. B. Jones, Toward tailoring the specificity of the SI pocket of subtilisin B. lentus chemical modification of mutant enzymes as a... 

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