Alanine activating enzyme

Heaton MP, Neuhaus FC. Biosynthesis of D-alanyl-lipoteichoic acid Cloning, nucleotide sequence, and expression of the LoctobadDus casei gene for the D-alanine-activating enzyme. J Bacteriol 1992 174 4707-4717. 

The molecular interaction between the activating enzyme and the amino acid is not known, but it probably varies with the type of activating enzyme. Some enzymes (. g., the alanine-activating enzyme) are unaffected by paramercuric benzoate, but others, like the tryptophan-activating enzyme, appear to be SH enzymes the activity of which depends on the presence of free SH groups in the molecule. Potassium ions are known to activate the tyrosine enzyme. 

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... 

The activating enzyme will also generate radicals from short peptides such as Arg-Val-Ser-Gly-Tyr-Ala-Val, which corresponds to residues 731-737 of the pyruvate formate-lyase active site. If Gly 734 is replaced by L-alanine, no radical is formed, but radical is formed if D-alanine is in this position. This suggests that the pro-S proton of Gly 734 is removed by the activating... 

EJJ Lugtenberg, A van Schijndel-vanDam. Temperature-sensitive mutants of Escherichia coli K-12 with low activities of the L-alanine adding enzyme and the D-alanyl-D-alanine adding enzyme. J Bacteriol 110 35 40, 1972. 

It is also possible to convert nonchiral readily available industrial organic chemicals into valuable chiral natural-analogue products. This is demonstrated by the conversion of achiral fumaric acid to L(-)-malic acid with fumarase as the active enzyme. The same compound is converted to the amino acid L(-h)-aspartic acid by Escherichia bacteria that contain the enzyme aspartase. If pseudomonas bacteria are added, another amino acid L-alanine is formed (Eq. 9.10). 

Nitrogen as a-amino nitrogen can be moved between carbon skeletons by a process known as transamination, requiring pyridoxal phosphate. Two important active enzymes for transamination are aspartate aminotransferase and alanine aminotransferase. In most transaminations, glutamate is either the donor of the amino group or the product of the transamination. 

In Escherichia coli. Salmonella typhimurium and Aerobacter aerogenes two soluble multi-activity enzymes or enzyme complexes function in the utilisation of chorismate (14) for L-phenyl-alanine and L-tyrosine synthesis An enzyme or enzyme complex (P-protein) containing chorismate mutase and prephenate dehydratase activities has been isolated and partially purified from Escherichia coli. Salmonella typhimurium and Aerobacter aerogenes. The enzyme complex catalyses the transformation of chorismate (14) to phenylpyruvate (32) and both enzymic activities are retained in physical association after chromatography on DEAE cellulose. Kinetic analysis indicated that in isolated enzyme systems direct synthesis of phenylpyruvate (32) from chorismate (14) does not occur. Prephenate (31) once formed dissociates from the enzyme surface and accumulates in the reaction medium. After a lag period it is converted to phenylpyruvate (32). Schmit, Artz and Zalkin also obtained evidence to show that functionally distinct sites (catalytic and regulatory) exist on the P-protein from Salmonella typhimurium for chorismate mutase and prephenate dehydratase activities. The P-protein was obtained from Escherichia coli K-12 by Davidson, Blackburn and Dopheide who showed that it existed in solution mainly as a dimer of similar (and probably identical) sub-units of... 

To the list of highly purified enzymes given in Table IV should be added an alanine-activatii enzyme obtained from hog liver cytoplasm by Webster SI), and a threonine-activating enzyme obtained from guinea pig liver by Allen et al. 36). 

---