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An elegant way to achieve a site-directed replacement of one or more amino acids in the protein is provided by genetically induced mutations into particular genes followed by expression of the mutant protein. This method was extensively used in the investigations of T. thermophilus EF-Tu. A compilation of mutants is presented in Tab. 19.3. In many cases the replacement of invariant and functionally important amino acids leads to the loss of the... 

Site-directed replacements of these residues, e.g., DION, E48Q, and D70N, result in almost a complete loss of activity (3). Replacements by Gly or Cys of the two conserved Asp residues of the MoLV RTase which correspond to Asp-10 and Asp-70 of the E. coli RNase HI also result in a 25- to 130-fold decrease in RNase H activity (52). In contrast, conservative substitutions in . coli RNase HI, i.e., DlOE, E48D, and D70E, result in mutant enzymes with 2-10% of the oi the wild type, while the remains invariant. Therefore, the carboxyl triad is involved in catalysis either directly or via bound Mg. The minor domain contains many basic residues whose side chains are mostly exposed to solvent. This feature implies that the minor domain is most likely involved in the binding to the polar region of the DNA RNA hybrids. 

Baum has reviewed the theory and the laboratory, rig, and plant experience which has led to the current views on concentration mechanisms. Essentially, interpretation is based on the difference between heat transfer at certain localised sites of the tube and that at the free surface. In normal boiling, the removal of steam from the surface leads to its direct replacement by liquid, so that accumulation of solutes does not occur to any appreciable extent. However, in certain cases, principally those three listed above, where there is an inadequate supply of water to the surface for boiling to be maintained, steam preferentially forms. Consequently, solute is deposited and accumulates with the formation of highly concentrated solution (lO -lO that in the bulk). 

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

The coordination of the Rieske cluster in the a subunit of benzene dioxygenase has been studied by site-directed mutagenesis. The replacement of His 98 or His 119 (corresponding to His 83/104 in NDO) by Cys resulted in a protein that was unable to coordinate a normal Rieske-type cluster (53). In the mutant His 98 Cys, a novel EPR spectrum with av = 1-94 was detected that is intermediate between... 

Fig. 14. Plot of the g values g,g ) and of the average g value g vs rhombicity (UJ of (a) wild type (open symbol) and variant forms (closed symbols) of the Rieske protein in yeast bci complex where the residues Ser 183 and Tyr 185 forming hydrogen bonds into the cluster have been replaced by site-directed mutagenesis [Denke et al. (35) Merbitz-Zahradnik, T. Link, T. A., manuscript in preparation] and of (b) the Rieske cluster in membranes of Rhodobacter capsulatus in different redox states of the quinone pool and with inhibitors added [data from Ding et al. (79)]. The solid lines represent linear fits to the data points the dashed lines reproduce the fits to the g values of all Rieske and Rieske-type proteins shown in Fig. 13.

The depurination of DNA, which happens spontaneously owing to the thermal lability of the purine N-glycosidic bond, occurs at a rate of 5000-10,000/cell/d at 37 °C. Specific enzymes recognize a depurinated site and replace the appropriate purine directly, without interruption of the phosphodiester backbone. 

His 118, Glu 146, His 128, His 142, Trpl, and His 14 as zinc binding ligands has been supported by a series of site-directed mutagenesis studies in which each of these residues was replaced with Ala each mutant bound only two zinc ions [35,64]. 

Replacement of Specific Residues By Site-directed Mutagenesis... 

Thus structural background suggests that the bound cyt c ccp adduct may actually consist of a distribution of structures. In this section, we consider the thermodynamics of binding cyt c and ccp, both for the native proteins, from different species, and proteins incorporating single site replacements, as prepared by site directed mutagenesis. 

The following replacements have been prepared by conventional or site directed mutagenesis ... 

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