Mutants histidine

Eraso, J.M. Kaplan, S. Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides a mutant histidine kinase. J. BacterioL, 177, 2695-2706 (1995)... 

In some cases, chemicals are known to be carcinogens from epidemiological studies of exposed humans. Animals are used to test for carcinogenicity, and the results can be extrapolated with some uncertainty to humans. The most broadly applicable test for potential carcinogens is the Bruce Ames procedure, which actually reveals mutagenicity. The principle of this method is the reversion of mutant histidine-requiring Salmonella bacteria back to a form that can synthesize their own histidine.12 The test is discussed in more detail in Section 8.4. 

Newmyer SL, Ortiz de Montellano PR (1995) Horseradish peroxidase His-42 -> Ala, His-42 -> Val, and Phe-41 -> Ala mutants. Histidine catalysis and control of substrate access to the heme iron. J Biol Chem 270 19430-19438... 

Vanderslice, R, Copeland, W. C., Robertus, J. D. (1986). Cloning and nucleotide sequence of wild type and a mutant histidine decarboxylase from Lactobacillus 30a. Journal of Biological Chemistry, 261, 15186-15191. 

The results of experiments in which the mutation was made were, however, a complete surprise. The Asp 189-Lys mutant was totally inactive with both Asp and Glu substrates. It was, as expected, also inactive toward Lys and Arg substrates. The mutant was, however, catalytically active with Phe and Tyr substrates, with the same low turnover number as wild-type trypsin. On the other hand, it showed a more than 5000-fold increase in kcat/f m with Leu substrates over wild type. The three-dimensional structure of this interesting mutant has not yet been determined, but the structure of a related mutant Asp 189-His shows the histidine side chain in an unexpected position, buried inside the protein. 

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]. 

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]. 

The behaviour of the mutant enzymes where, for example, histidine-152 has been changed to alanine is compared with that of wild type enzymes.60 The 31P NMR chemical shift values and signal width for H152A mutant enzyme have shown the presence of two conformers open and closed forms of the enzyme that interconvert slowly on the NMR time scale. The tightness of the binding of the cofactor to the protein surface and its protonation state have been also discussed for intermediate Schiff bases in different steps of the catalytic cycle (Table 1). 

Bricker, T. M, J. Morvant, N. Masri, H. M. Sutton, L. K. Frankel (1998) Isolation of a highly active photosystem n preparation from Synechocystis 6803 using a histidine tagged mutant of CP 47. Biochim. Biophys. Acta, 1409 50-57... 

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