Activity alanine, hydrolysis

Most of the acids obtained from proteins by hydrolysis with acids are optically active alkaline hydrolysis yields racemic forms. Fischer has prepared many optically active peptids, and has discovered that the configuration of the molecule is an important factor in its hydrolysis by trypsin. For example, d-alanyl-d-alanine is hydrolyzed, but d-alanyl-i-alanine is not. Racemic peptids, that is, those which consist of a mixture of equal parts of dextro and levo compounds, are hydrolyzed asymmetrically, only one form, and that the form which occurs in proteins, being attacked by the enzyme. Up to the present, only those peptids have been hydrolyzed which contain the optically active forms of the amino-acids which result from the hydrolysis of naturally occurring compounds. 

The enzymes are predominantly secreted in the fundus. The general proteolytic activity shows an optimum of about pH 1.8 to 2.2. Low activity towards hydrolysis of carbobenzoxy-tyrosyl-alanine and high activity towards acetyl-phenylalanyl-diiodotyrosine has been reported as characteristic for pepsin A (20). By electrophoresis at pH 8.5 for pepsinogen, or pH 5.0 to 5.4 for pepsin, this group of enzymes has the greatest mobility toward the anode (21-25). 

Some of these enzymes are mainly secreted in the antropyloric part of the stomach. Names like pepsin B, C, gastricsin, or pyloric pepsin have been used for members of this group. Interspecies relationship between these enzymes is not yet clear. The general proteolytic activity shows a maximum of about pH 3.0 (17,19). Low activity towards hydrolysis of acetyl-phenylalanyl-diiodotyrosine and high activity towards carbobenzoxy-tyrosyl-alanine have been reported as characteristic for both pepsin C and gastricsin (20) the two designations most likely describe the same enzyme. A similar relation between the activities toward the two substrates has been found for bovine pepsin B (M. K. Harboe, personal communication). ... 

The sequence of each different peptide or protein is important for understanding the activity of peptides and proteins and for enabling their independent synthesis, since the natural ones may be difficult to obtain in small quantities. To obtain the sequence, the numbers of each type of amino acid are determined by breaking down the protein into its individual amino acids using concentrated acid (hydrolysis). For example, hydrolysis of the tetrapeptide shown in Figure 45.3 would give one unit of glycine, two units of alanine, and one unit of phenylalanine. Of course, information as to which amino acid was linked to which others is lost. 

The trienomycins ate isolated from Streptomjces sp. 83-16 (43,44). The assigned stmctures (Fig. 12) were based on spectral data. Acid hydrolysis of trienomycin A yielded D-alanine (42,44). The trienomycins have no antimicrobial activity but have good antitumor activity. Trienomycin A is the most active, exhibiting good in vivo antitumor activity against sarcoma 180 and P 388 leukemia in mice (241). 

The ansatrienins ate produced by Streptomjces collinus Tb 1982. The stmctures (Fig. 12) were assigned on the basis of spectral data of the intact antibiotics as well as several derivatives. Acid hydrolysis of the ansatrienins yields L-alanine. The stmctures of ansatrienins A and B ate the same as those for mycotrienins 1 and 11, respectively, except for the configuration of the alanine moiety. The ansatrienins ate active against fungi (45,46). 

A proline derived chiral nickel complex 1 may be used instead of oe,/J-unsaturated esters of lactones modified with a chiral alcohol as the Michael acceptor. The a,(9-unsaturated acid moiety in 1 reacts with various enolates to afford complexes 2 with diastereomcric ratios of 85 15 to 95 5. Hydrolysis of the imine moiety yields the optically active /(-substituted r-alanines. A typical example is shown296. 

The phosphotriesterase from Pseudomonas diminuta was shown to catalyze the enantioselective hydrolysis of several racemic phosphates (21), the Sp isomer reacting faster than the Rp compound [65,66]. Further improvements using directed evolution were achieved by first carrying out a restricted alanine-scan [67] (i.e. at predetermined amino acid positions alanine was introduced). Whenever an effect on activity/ enantioselectivity was observed, the position was defined as a hot spot. Subsequently, randomization at several hot spots was performed, which led to the identification of several highly (S)- or (R)-selective mutants [66]. A similar procedure was applied to the generation of mutant phosphotriesterases as catalysts in the kinetic resolution of racemic phosphonates [68]. 

Amino acid esters act as chelates to Co111 for example, the /3-alanine isopropyl ester is known as both a chelate and as an /V-bonded monodentate,983 and the mechanism of hydrolysis of the ester, which is activated by coordination, to yield chelated /3-alanine has been closely examined. 

N-Carbobenzoxy-L-alanine-/>-nitrophenyl ester is a specific substrate for elastase in which the rate-limiting step is deacylation, that is, hydrolysis of the acyl-enzyme intermediate. In 70% methanol over a reasonable temperature range the energy of activation of the turnover reaction, that is, deacylation, is 15.4 kcal mol. In the pH 6-7 region in this cryoprotective solvent, the turnover reacdon can be made negligibly slow at temperatures of -50 C or below. Under such conditions/i-nitro-phenol is released concurrent to acyl enzyme formation in a 1 1 stoichiometry with active enzyme in the presence of excess substrate. In other words, even at low temperatures, the acylation rate is much faster than deacylation and the acyl enzyme will accumulate on the enzyme. The rate of acyl-enzyme formation can be monitored by following the rate of p-nitrophenol release, and thus the concentration of trapped acyl enzyme may be determined. This calculadon has been carried out and... 

This enzyme [EC 3.4.16.4], also known as serine-type D-alanyl-D-alanine carboxypeptidase, catalyzes the hydrolysis of D-alanyl-D-alanine to yield two D-alanine. This enzyme comprises a group of membrane-bound, bacterial enzymes of the peptidase family Sll. They are distinct from the zinc D-alanyl-D-alanine carboxypeptidase [EC 3.4.17.14]. The enzyme also hydrolyzes the D-alanyl-D-alanine peptide bond in the polypeptide of the cell wall. In addition, the enzyme will also catalyze the transpeptidation of peptidyl-alanyl moieties that are A-acetyl-substituents of D-alanine. The protein is inhibited by j8-lactam antibiotics, which acylate the active-site seryl residue. 

Both enzymes are inhibited by sodium borohydride and also by nitromethane. After reduction with NaB3H4 and hydrolysis, 3H-containing alanine was isolated. This suggested that they contain dehydroalanine, which could arise by dehydration of a specific serine residue.286,287 For phenylalanine ammonia-lyase from Pseudomonas putida this active site residue has been identified as S143. Replacement by cysteine in the S143C mutant also gave active enzyme while S143A... 

Wild-type subtilisin BPN with the mutation Ser — Cys-24 has a kcat value of 59 s 1 and a KM value of 200 fxM with the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, compared with a rate constant of 1.1 X 10 8 s 1 for its spontaneous hydrolysis under the same conditions. Replacement of Asp-32, His-64, and Ser-221 one at a time by alanine reduced the value of kcat by factors of 3 X 104, 2 X 106, and 2 X 106, respectively. Converting all three to alanine also decreases activity by 2 X 106. The value of KM increases only by a factor of two on all these mutations.34 It is unlikely that the residual activity results from the presence of a small amount of wild-type active site in the thiol mutants... 

The alanine racemization catalyzed by alanine racemase is considered to be initiated by the transaldimination (Fig. 8.5).26) In this step, PLP bound to the active-site lysine residue forms the external Schiff base with a substrate alanine (Fig. 8.5, 1). The following a-proton abstraction produces the resonance-stabilized carbanion intermediates (Fig. 8.5, 2). If the reprotonation occurs on the opposite face of the substrate-PLP complex on which the proton-abstraction proceeds, the antipodal aldimine is formed (Fig. 8.5,3). The subsequent hydrolysis of the aldimine complex gives the isomerized alanine and PLP-form racemase. The random return of hydrogen to the carbanion intermediate is the distinguishing feature that differentiates racemization from reactions catalyzed by other pyridoxal enzymes such as transaminases. Transaminases catalyze the transfer of amino group between amino acid and keto acid, and the reaction is initiated by the transaldimination, followed by the a-proton abstraction from the substrate-PLP aldimine to form a resonance-stabilized carbanion. This step is common to racemases and transaminases. However, in the transamination the abstracted proton is then tranferred to C4 carbon of PLP in a highly stereospecific manner The re-protonation occurs on the same face of the PLP-substrate aldimine on which the a-proton is abstracted. With only a few exceptions,27,28) each step of pyridoxal enzymes-catalyzed reaction proceeds on only one side of the planar PLP-substrate complex. However, in the amino acid racemase... 

A Wittig reaction, for instance, transforms 30a in 30b M. Thus, acylation of 22 followed by Wittig reactions and hydrolysis offers the possibility to prepare a variety of optically active a-alkenyl alanines of type 28. 

---