Site-specific mutants

One example of a sequence determinant of redox potentials that has been identified in this manner is an Ala-to-Val mutation at residue 44, which causes a 50 mV decrease in redox potential (and vice versa) in the rubredoxins [68]. The mutation was identified because the sum of the backbone contributions to ( ) of residues 43 and 44 change by 40 mV due to an —0.5 A backbone shift away from the redox site. This example points out the importance of examining the backbone contributions. The corresponding site-specific mutants have confirmed both the redox potential shift [75] and the structural shift [75]. 

A second example is that of an Ala-to-Cys mutation, which causes the fonnation of a rare SH S hydrogen bond between the cysteine and a redox site sulfur and a 50 mV decrease in redox potential (and vice versa) in the bacterial ferredoxins [73]. Here, the side chain contribution of the cysteine is significant however, a backbone shift can also contribute depending on whether the nearby residues allow it to happen. Site-specific mutants have confirmed the redox potential shift [76,77] and the side chain conformation of cysteine but not the backbone shift in the case with crystal structures of both the native and mutant species [78] the latter can be attributed to the specific sequence of the ferre-doxin studied [73]. 

C. K. Tu, D. N. Silverman, C. Forsman, B. H. Jonsson, S. Lindskog, Role of Histidine 64 in the Catalytic Mechanism of Human Carbonic Anhydrase II Studied with a Site-Specific Mutant , Biochemistry 1989, 28, 7913-7918. 

L. T. Laughlin, H. F. Tzeng, S. Lin, R. N. Armstrong, Mechanism of Microsomal Epoxide Hydrolase. Semifunctional Site-Specific Mutants Affecting the Alkylation Half-Reaction , Biochemistry 1998, 37, 2897 - 2904. 

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). 

Abstract. Walter Kauzmann stated in a review of protein thermodynamics that volume and enthalpy changes are equally fundamental properties of the unfolding process, and no model can be considered acceptable unless it accounts for the entire thermodynamic behaviour (Nature 325 763-764, 1987). While the thermodynamic basis for pressure effects has been known for some time, the molecular mechanisms have remained rather mysterious. We, and others in the rather small field of pressure effects on protein structure and stability, have attempted since that time to clarify the molecular and physical basis for the changes in volume that accompany protein conformational transitions, and hence to explain pressure effects on proteins. The combination of many years of work on a model system, staphylococcal nuclease and its large numbers of site-specific mutants, and the rather new pressure perturbation calorimetry approach has provided for the first time a fundamental qualitative understanding of AV of unfolding, the quantitative basis of which remains the goal of current work. 

Fig. 9.2. Ribbon diagram of Snase, PDB 1EYO [13]. The single tryptophan is shown in dark gray and one of the residues for which a number of site specific mutants has been studied, valine 66, is shown in black...

Over the years, a very large amount of static and time-resolved spectroscopy of various kinds, as well as studies of site-specific mutants of bacteriorhodopsin, has generated kinetic models for the transport cycle ( photocycle ) and identified the side chains of importance (Haupts et al., 1999 Lanyi and Varo, 1995 Oesterhelt, 1998). The results had begun to identify the molecular events that underlie the interconversions of the spectroscopically distinct intermediate states termed J, K, L, M, N, and O. Together with low-resolution 3D maps of the protein and some of the intermediate states from cryoelectron microscopy of 2D crystals, these results suggested the beginnings of a mechanistic model... 

Although the ET processes represented by reactions 3 and 5 are nonphysiologi-cal, they can provide useful information. Thus, by varying the structure of the flavin it is possible to probe the steric and electrostatic environment of a protein redox center [38, 39]. This can be used as a method for assessing functional relationships between members of a homologous series of redox proteins (kinetic taxonomy). It is also possible to use these reactions to test the functional integrity of a redox center in a site-specific mutant [43]. 

Protease mutants were prepared, which showed higher catalytic activity for the enzymatic polymerization of amino acid esters in an aqueous DMF solution. The molecular weight greatly increased by using a subtilisin mutant (subtilisin 8350) derived from BPN (subtilisin from Bacillus amyloliquefa-ciens) via six site-specific mutants (Met 50 Phe, Gly 169 Ala, Asn 76 Asp, Gin 206 Cys, Tyr 217 Lys, and Asp 218 Ser) in the polymerization of L-methionine methyl ester in the aqueous DMF.240 Another mutant (subtilisin 8397), which is the same as 8350 without... 

Several reaction mechanisms, alternative to the retro-aldol cleavage, have been proposed on the basis of kinetic, stereochemical, and reaction specificity studies and exhaustively reviewed. However, only the relatively recent resolution of crystal structures of the enzyme from several sources " and characterization of site-specific mutants have resolved some of the crucial questions concerning the catalytic mechanism of SHMT. 

PHA synthases are classified into the afl-hydrolase super-family and they are structurally similar to lipases, which are the typical members of this superfamily. On the basis of homology search, all PHA synthases contain the ap-hydrolase domain at the C-teiminal region of PhaC. Many biochemical studies have been extensively performed on type I, II, and III PHA synthases with site-specific mutants. " However, unfortunately, the tertiary structure of PHA synthases has not yet been resolved by X-ray dififaction analysis due to the difficulty in crystallization of PHA synthase. Although several tertiary stmcture models of PHA synthases have been proposed, based on the homology with crystallographically solved lipases, they are of limited value for the creation of improved or new enzymes. 

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