Catalytic base

Aleshin and coworkers (49) have reported the X-ray crystal structure at 2.2-A resolution of a G2-type variant produced by Aspergillus awamori. Meanwhile, an attempt was made to determine the amino acid residues that participate in the substrate binding and catalysis provided by G2 of A. niger (52). The results of the chemical approach indicated that the Asp-176, Glu-179, and Glu-180 form an acidic cluster crucial to the functioning of the enzyme. This conclusion was then tested by site-specific mutagenesis of these amino acid residues, which were replaced, one at a time, with Asn, Gin, and Gin, respectively (53). The substitution at Glu-179 provided an inactive protein. The other two substitutions affected the kinetic parameters but were not of crucial importance to the maintenance of activity. The crystal structure (49) supports the conclusion that Glu-179 functions as the catalytic acid but Asp-17 6 does not appear to be a good candidate for provision of catalytic base. Thus, there still exists considerable uncertainty as to how the disaccharide is accepted into the combining site for hydrolysis. Nevertheless, the kind of scheme presented by Svensson and coworkers (52) almost surely prevails. 

The Baylis-Hillman reaction (Scheme 3) of ethyl vinyl ketone with electron-deficient aromatic aldehydes (e.g. where R = 0-NO2C6H4), in MeCN or EtCN solution, has been found to proceed enantioselectively in presence of catalytic base (32) derived from proline. The Michael adduct formed between the catalyst and the vinyl... 

Building on Inoue s pioneering work and using Andersson s versatile diamine, Kozmin and coworkers have described a catalytic base-induced isomerization of silacyclopentene oxide 139 [Eq. (10.41)] as a potential route to acyclic polyol domains, important components of a number of natural products. Epoxida-tion of 140, epoxide ring opening, and oxidation of the C—Si bonds provides tetra-ols in good overall yield and selectivity ... 

W. P. Burmeister, S. Cottaz, P. Rollin, A. Vasella, and B. Henrissat, High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base,./. Biol. Chem., 275 (2000) 39385-39393. 

The active site of enoyl-CoA isomerase is a good example of an active site built on the framework of the crotonase fold. It is now weU established that this crotonase fold provides an active site framework that has been used by Nature for a wide range of different chemical reactions, as reviewed recently [73, 85]. The reaction of this enoyl-CoA isomerase is initiated by a catalytic base, Glul36, abstracting a proton from the Ca-carbon, generating the negatively charged enolate... 

Figure 4.5 The active site of enoyl-CoA isomerase, complexed with octanoyl-CoA (PDB 1SC4). In this structure the oxygen atom of Clul36 which acts as the catalytic base points to the C2 carbon of the ligand and is therefore well positioned for proton...

The catalytic center is formed by residues from both lobes. Sequence comparisons, mutation experiments and biochemical studies indicate an essential fimction in catalysis of phosphate transfer for the conserved amino acids Lys72, Aspl66 and Aspl84 (numbering of PKA). However, the catalytic mechanism of phosphate transfer is not definitely established. It is generally assumed that Aspl66, which is invariant in all protein kinases, serves as a catalytic base for activation of the Ser/Thr hydroxyl and that the reaction takes place by an in-line attack of the Ser-OH at the y-phosphate. 

Nondek et al. (46) reported an innovative approach to the analysis of N-methylcarbamates in river water using postcolumn reaction detection. Separation of the underivatized N-methylcarbamates was carried out on a reversed-phase column hooked directly to a bed reactor packed with Aminex A-28, a tetraalkylammonium anion-exchange resin. The packed bed catalytically base-hydrolyzed the carbamates and... 

The catalytic base B might be HO or a weaker base such as ammonia or even water. For reactions the rate is proportional only to the concentration of OH and the presence of other weaker bases has no effect.129b Such catalysis is referred to as specific hydroxyl ion catalysis.19 More commonly, the rate is found to depend both on [OH ] and on the concentration of other weaker bases. In such cases the apparent first-order rate constant (fcobs) for the process is represented by a sum of terms (Eq. 9-88). The term kH2, ) is the rate in... 

For example, if either the histidine or the serine of the triad of subtilisin was replaced by alanine the catalytic activity decreased by a factor of 2 x 106 and replacement of the aspartate of the triad by alanine decreased activity by a factor of 3 x 104.229/258 When Asp 102 of trypsin is replaced by asparagine the catalytic activity falls by four orders of magnitude.259 This may be in part because the histidine in this mutant is hydrogen bonded to Asn 102 as the tautomer with a proton on Ne, the nitrogen that should serve as the catalytic base in step b (Fig. 12-11).260 A mutant in which Ser 214 (see Fig. 12-10) was replaced with alanine is fully active but charged residues in this position interfere with catalysis.261... 

Various bacterial ribonucleases as well as the fungal ribonucleases Ty Uj, and U2 (see also Fig. 5-43) have amino acid sequences related to that of RNase A763 764 764a but with distinctly different three-dimensional structures. The active sites contain Glu, His, and Arg side chains. For RNase Ty Glu 58 and His 92 appear to provide acid-base catalysis with assistance from Tyr 38, Arg 77, and His 40.763 765 A glutamate carboxylate also appears to be the catalytic base in the related RNase, called barnase, from Bacillus amyloliquefaciens.766... 

A relative of the kinases is adenylate cyclase, whose role in forming the allosteric effector 3, 5 -cyclic AMP (cAMP) was considered in Chapter 11. This enzyme catalyzes a displacement on Pa of ATP by the 3 -hydroxyl group of its ribose ring (see Eq. 11-8, step a). The structure of the active site is known.905 Studies with ATPaS suggest an in-line mechanism resembling that of ribonuclease (step a, Eq. 12-25). However, it is Mg2+ dependent, does not utilize the two-histidine mechanism of ribonuclease A, and involves an aspartate carboxylate as catalytic base.906 All isoforms of adenylate cyclase are activated by the a subunits of some G proteins (Chapter 11). The structures907 of Gsa and of its complex with adenylate kinase905 have been determined. The Gsa activator appears to serve as an allosteric effector. 

A closely related E. coli protein is a 79-kDa multifunctional enzyme that catalyzes four different reactions of fatty acid oxidation (Chapter 17). The amino-terminal region contains the enoyl hydratase activity.32 A quite different enzyme catalyzes dehydration of thioesters of (3-hydroxyacids such as 3-hydroxydecanoyl-acyl carrier protein (see Eq. 21-2) to both form and isomerize enoyl-ACP derivatives during synthesis of unsaturated fatty acids by E. coli. Again, a glutamate side chain is the catalytic base but an imidazole group of histidine has also been implicated.33 This enzyme is inhibited irreversibly by the N-acetylcysteamine thioester of 3-decynoic acids (Eq. 13-8). This was one of the first enzyme-activated inhibitors to be studied.34... 

Figure 13-2 View of the active site of yeast enolase containing a bound molecule of 2-phospho-D-glycerate. The catalytic magnesium ion is at the left but the "conformational" metal is not visible here. The imidazole group of His 159 serves as the catalytic base and the -NH3+ of Lys 396 or Lys 34573b as the catalytic acid. From Vinarov and Nowak.69...

The degradation of mandelic acid by the bacterium Pseudomonas putida (Chapter 25) is initiated by mandalate racemase, another (a/(3)8-barrel protein.101 X-ray structures of bound inhibitors together with modeling suggest that the side chain of Lys 264 is the catalytic base that abstracts the a-H from S-mandelate (Fig. 13-5) and that the catalytic pair of His 297 and Asp 270 acts as proton donor, or, in the reverse direction, as catalytic... 

Mechanisms. Studies of model reactions473-476 and of electronic, Raman,456 477 478 ESR,479/480 and NMR spectra and kinetics481 have contributed to an understanding of these enzymes.459 461 464 482 483 For these copper amine oxidases the experimental evidence suggests an aminotransferase mechanism.450 453 474 4743 d Tire structure of the E.coli oxidase shows that a single copper ion is bound by three histidine imidazoles and is located adjacent to the TPQ (Eq. 15-53). Asp 383 is a conserved residue that may be the catalytic base in Eq. 15-53.474b A similar mechanism can be invoked for LTQ and TTQ. 

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