Protein Engineering Studies

Saccharomyces cerevisiae is quite commonly used as a host for expression of proteins from cloned genes from yeasts and other organisms. Because flavocytochrome 62 is a yeast enzyme and procedures for isolation of intact enzyme from yeast had already been developed, Reid et al. (143) developed a system for expression of S. cerevisiae flavocy- 

coli harboring a plasmid, pDS-b2 (Fig. 16), designed for in vitro transcription and translation, were noticeably pink in color 147). This results from constitutive expression of flavocytochrome 62 at up to 5% of the total soluble protein in these cells. Expressed enzyme contains full stoichiometric amounts of FMN and heme 147). Expression of flavocytochrome 62 in vivo was not expected with this vector because there is no E. coli ribosome-binding site (rbs). It appears that a fortuitous rbs exists within the region of DNA encoding the mitochondrial targeting sequence and this leads to initiation of translation at Met 6 of the mature flavocytochrome 62 147). The absence of residues 1-5 

Comparison of Kinetic Properties for Oxidation OF l-Lacate by Wied-Tvpe S. cerevisiae Flavocytochrome 62  

Several point mutations in the S. cerevisiae flavocytochrome coding sequence have been constructed by oligonucleotide-directed site-specific mutagenesis of the cloned gene (143). The enzyme-coding region was transferred to a plasmid designed both for expression of 

Each of the mutant forms of flavocytochrome 62 constructed in our laboratory has been purified and subjected to steady-state kinetic analysis. This simple first approach has often given us, very quickly, an impression of the mechanistic effects of the mutation, but perhaps more importantly has dictated which mutant enzymes deserved further characterization and in which way they should be analyzed. 

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Many enzymes have been the subject of protein engineering studies, including several that are important in medicine and industry, eg, lysozyme, trypsin, and cytochrome P450. SubtiHsin, a bacterial serine protease used in detergents, foods, and the manufacture of leather goods, has been particularly well studied (68). This emphasis is in part owing to the wealth of stmctural and mechanistic information that is available for this enzyme. 

With subtihsin nature has provided us with a model system for protein engineering studies (Wells and Estell, 1988). Being a small single domain serine protease... 

A related phenomenon is half-of-the-sites or half-site reactivity, by which an enzyme containing 2n sites reacts (rapidly) at only n of them (Table 10.2). This can be detected only by pre-steady state kinetics. The tyrosyl-tRNA synthetase provides a good example, in that it forms 1 mol of enzyme-bound tyrosyl adenylate with a rate constant of 18 s1, but the second site reacts 104 times more slowly.13 However, as will be seen in Chapter 15, section J2b, protein engineering studies on the tyrosyl-tRNA synthetase unmasked a pre-existing asymmetry of the enzyme in solution. 

The prerequisite for protein engineering studies is that the enzyme has been cloned and expressed. Further, unless only relatively crude information is required, it is essential that the structure has been solved at high resolution. Accurate structure-activity studies require even more stringent criteria absolute values of rate constants. The two following procedures, which were discussed earlier (Chapter 4, section E), must be available. Both depend on the accumulation of an enzyme-bound intermediate or product on the reaction pathway. 

The more recent structural evidence supports the role of substrate distortion. A trisaccharide in the BCD sites of crystalline hen egg white lysozyme is distorted to the half-chair,229 as is a substrate bound to the Asp-52 — Ser mutant studied in solution by NMR.230 Protein engineering studies on the residues involved support the conclusions.231 A chitobiose bound to a crystalline chitobiase has the equivalent sugar distorted to a sofa.232... 

There is a severe practical problem in looking for correlations between the rate constants for folding of small proteins and their structural or thermodynamic properties—specific structural features can dominate the rate of folding. For example, we know from the protein engineering studies on barnase and CI2 that specific mutations can slow down the rate of folding by several orders of... 

Ultimate goal of DFS studies is to create an energy landscape of the protein which would serve as blue print for rational protein engineering studies.15,16 Reconstituting... 

Protein engineering studies have allowed modulation of D-amino acid oxidases oligomerization state, stability (which is significantly increased in the immobihzed form), FAD binding, and substrate specificity (for a review see [57]). The residue... 

E. coli class I RNR was evident already in 1983, long before the three-dimensional structures were known or protein engineering studies had been adopted. A common net result of ineubations with 2 -substituted substrate analogues is inactivation of the R2 eomponent by loss of the tyrosyl radical, and formation of a transient radieal loealised to the active site of the enzyme which eventually leads to inaetivation of protein R1 by covalent modification. This effective dual inhibition of RNR by some of the 2 -substituted substrate analogues is eurrently explored in antiproliferative treatment in clinical trials (Nocentini, 1996). 

The third example in this chapter is about cellulase, Cel5A from Acidothermus cellulolyticus [69], Its crystal structure complexed with a natural substrate cellote-traose was resolved in 1996. Since then, the protein engineering study around this enzyme has attracted much attention. Especially, one of its mutants, Y245G, was found to increase the catalytic activity about 20 %, when combined with Cel7A. Indeed, one of the bottlenecks for effective biomass conversion is the low catalytic ability of cellulases. Theoretical investigations were carried out to understand the catalytic process using the hybrid QM/MM scheme. 

Taken together, lipocalins provide attractive candidates in order to engineer novel ligand specificities. Features hke their small size (typically between 150 and 180 residues), monomeric polypeptide composition, dispensable posttranslational modification, and robust protein fold not only facihtate protein-engineering studies but also provide advantages for practical apphcations. 

Protein Engineering Studies Providing a Rational Explanation for Enzyme Specificity... 

Mammalian cells produce two t-PA variants of N-linked glycosylation, type 1 (at asparagines 117,184, and 448) and type 2 (only as asparagines 117 and 448). The rate of fibrin-dependent plasminogen activation is two- to threefold faster for type 2 compared with type 1. The cDNA obtained from a human melanoma cell line was expressed in CHO cells to achieve glycosylation and a protein identical to the natural protein. Protein engineering studies have produced variant t-PA molecules with modified pharmacokinetics, affinity tor fibrin, catalytic activity, and side effects. 

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