Mutants, active site

The Protein Data Bank PDB ID 1A71 Colby T D Bahnson B J Chin J K Klinman J P Goldstein B M Active Site Modifications m a Double Mutant of Liver Alcohol Dehydrogenase Structural Studies of Two Enzyme Ligand Com plexes To be published... 

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

Scheme 10.8 Biosynthesis of epothilone. Individual PKS domains are represented as circles and individual NRPS domains as hexagons. Acyl carrier proteins (ACPs) and thiola-tion domains (T) are posttranslationally modified by a phos-phopantetheinyl group to which the biosynthetic intermediates are covalently bound throughout the chain assembly. The thioesterase domain (TE) cyclizes the fully assembled carbon chain to give the 16-membered lactone. Following dehydration of Cl 2—Cl 3 to give epothilones C and D, the final step in epothilone biosynthesis is the epoxidation of the C12=C13 double bond by the cytochrome P450 enzyme P450epol<. KS ketosyn-thase KS(Y) active-site tyrosine mutant of KS AT acyltransfer-ase C condensation domain A adenylation domain ...

The lipase (PAL) used in these studies is a hydrolase having the usual catalytic triad composed of aspartate, histidine, and serine [42] (Figure 2.6). Stereoselectivity is determined in the first step, which involves the formation of the oxyanion. Unfortunately, X-ray structural characterization of the (S)- and (J )-selective mutants are not available. However, consideration of the crystal structure of the WT lipase [42] is in itself illuminating. Surprisingly, it turned out that many of the mutants have amino acid exchanges remote from the active site [8,22,40]. 

The ANEH-mutant displaying enhanced enantioselectivity ( =10.8) was sequenced and shown to be characterized by three mutations, A217V near the active site and K332E and A390E both at remote positions [58]. The X-ray crystal structure of the WT ANEH had been analyzed earlier [61], revealing a dimer comprising identical... 

In another study that appeared prior to the advent of CASTing, the traditional combination of epPCR and DNA shuffling was used to enhance the enantioselectivity of the hydrolytic kinetic resolution of p-nitro phenyl glycidyl ether and other epoxides catalyzed by the EH from Agrobacterium radiobacter [59]. Several mutants were obtained with up to 13-fold improved enantioselectivity. The amino acid exchanges took place around the active site. 

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

This model clearly shows that the catalytic machinery involves a dyad of histidine and aspartate together with the oxyanion hole. Hence, it does not involve serine, which is the key amino acid in the hydrolytic activity of lipases, and, together with aspartate and histidine, constitutes the active site catalytic triad. This has been confirmed by constructing a mutant in which serine was replaced with alanine (Serl05Ala), and finding that it catalyzes the Michael additions even more efficiently than the wild-type enzyme (an example of induced catalytic promiscuity ) [105]. 

Both enzymes belong to the family of a,p-hydrolases." The active site of MeHNL is located inside the protein and connected to the outside through a small channel, which is covered by the bulky amino acid tryptophane 128." It was possible to obtain the crystal structure of the complex with the natural substrate acetone cyanohydrin with the mutant SerSOAla of MeHNL. This complex allowed the determination of the mode of substrate binding in the active site." A summary of 3D structures of known HNLs was published recently." " ... 

The CD spectrum of the C188S mutant is essentially the same as that of the wild-type enzyme, which reflects that the tertiary structure of this mutant changed little compared to that of the wild-type enzyme. Calculation of the content of secondary structure of the mutant enzyme based on J-600S Secondary Structure Estimation system (JASCO) also showed that there is no significant change in the secondary structure of the mutant. The fact that the k value of this mutant is extremely small despite little change in conformation clearly indicates that Cysl88 is located in the active site. 

TKase is a homodimeric protein with a subunit of about 70kDa. The X-ray structures of TKase of E. colif S. cerevisiaeX Leishmania mexicana and mize have been solved. In addition, the crystal structures of a number of site-directed mutants have been determined. Schneider and co-workers have reported a series of studies in which they have mutated important residues of active site of TKase to elucidate the reaction mechanism and explain the origin of the stereospecificity of the C—C bond-forming process (Table The conserved... 

Renirie R, W Hemrika, R Wever (2000) Peroxidase and phosphatase activity of active-site mutants of vanadium chloroperoxidase from the fungus Curvularia inaequalis. J Biol Chem 275 11650-11657. 

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