Pyridoxal-5-Phosphate hydrolysis

An example of a biologically important aide hyde is pyridoxal phosphate which is the active form of vitamin Bg and a coenzyme for many of the reac tions of a ammo acids In these reactions the ammo acid binds to the coenzyme by reacting with it to form an imine of the kind shown in the equation Re actions then take place at the ammo acid portion of the imine modifying the ammo acid In the last step enzyme catalyzed hydrolysis cleaves the imme to pyridoxal and the modified ammo acid... 

The mechanism of the first part of transamination is shown in Figure 29.14. The process begins with reaction between the a-amino acid and pyridoxal phosphate, which is covalently bonded to the aminotransferase by an iminc linkage between the side-chain -NTI2 group of a lysine residue and the PLP aldehyde group. Deprotonation/reprotonation of the PLP-amino acid imine in steps 2 and 3 effects tautomerization of the imine C=N bond, and hydrolysis of the tautomerized imine in step 4 gives an -keto acid plus pyridoxamine... 

Novozymes, a subtilisin produced by Bacillus licheniformis, was used by Chen et al ° to carry out a dynamic kinetic resolution of benzyl, butyl, or propyl esters of DL-phenylalanine, tyrosine, and leucine. The hydrolysis was performed at pH 8.5 in 2-methyl-2-propanol/water (19 1) and the freed L-amino acids precipitated. The key feature bringing about continual racemization of the remaining D-amino acid esters was the inclusion of 20 mmol 1 pyridoxal phosphate. 

This pyridoxal-phosphate-dependent enzyme [EC 4.4.1.8], also referred to as /3-cystathionase and cystine lyase, catalyzes the hydrolysis of cystathionine to yield homocysteine, pyruvate, and ammonia. 

This pyridoxal-phosphate-dependent enzyme [EC 3.7.1.3] catalyzes the hydrolysis of kynurenine to produce anthranilate and alanine. 3 -Hydroxykynurenine and some other (3-arylcarbonyl)alanines can also be acted upon by this enzyme. 

L-Serine dehydratase [EC 4.2.1.13], also known as serine deaminase and L-hydroxyaminoacid dehydratase, catalyzes the pyridoxal-phosphate-dependent hydrolysis of L-serine to produce pyruvate, ammonia, and water. In a number of organisms, this reaction is also catalyzed by threonine dehydratase. 

This enzyme [EC 4.1.99.1], also known as L-tryptophan indole-lyase, catalyzes the hydrolysis of L-tryptophan to generate indole, pyruvate, and ammonia. The reaction requires pyridoxal phosphate and potassium ions. The enzyme can also catalyze the synthesis of tryptophan from indole and serine as well as catalyze 2,3-elimination and j8-replacement reactions of some indole-substituted tryptophan analogs of L-cysteine, L-serine, and other 3-substituted amino acids. 

The synthesis pathway of quinolizidine alkaloids is based on lysine conversion by enzymatic activity to cadaverine in exactly the same way as in the case of piperidine alkaloids. Certainly, in the relatively rich literature which attempts to explain quinolizidine alkaloid synthesis °, there are different experimental variants of this conversion. According to new experimental data, the conversion is achieved by coenzyme PLP (pyridoxal phosphate) activity, when the lysine is CO2 reduced. From cadeverine, via the activity of the diamine oxidase, Schiff base formation and four minor reactions (Aldol-type reaction, hydrolysis of imine to aldehyde/amine, oxidative reaction and again Schiff base formation), the pathway is divided into two directions. The subway synthesizes (—)-lupinine by two reductive steps, and the main synthesis stream goes via the Schiff base formation and coupling to the compound substrate, from which again the synthetic pathway divides to form (+)-lupanine synthesis and (—)-sparteine synthesis. From (—)-sparteine, the route by conversion to (+)-cytisine synthesis is open (Figure 51). Cytisine is an alkaloid with the pyridone nucleus. 

Effective concentration 65-72 entropy and 68-72 in general-acid-base catalysis 66 in nucleophilic catalysis 66 Elastase 26-30, 40 acylenzyme 27, 40 binding energies of subsites 356, 357 binding site 26-30 kinetic constants for peptide hydrolysis 357 specificity 27 Electrophiles 276 Electrophilic catalysis 61 metal ions 74-77 pyridoxal phosphate 79-82 Schiff bases 77-82 thiamine pyrophosphate 82-84 Electrostatic catalysis 61, 73, 74,498 Electrostatic effects on enzyme-substrate association rates 159-161... 

The covalent intermediates can be attacked by a second nucleophile to cause the release of the product. When the second nucleophile is water, the overall reaction is called hydrolysis. Also, in many cases the nucleophile is not simply an amino acid side chain of the enzyme but a prosthetic group an example is pyridoxal phosphate in the transaminases (Chap. 15). 

ALA synthase is a pyridoxal phosphate-dependent enzyme and promotes Schiff-base formation between its coenzyme and glycine (67 in Fig. 37). Nucleophilicity at C-2 of the glycine could be generated either by decarboxylation or by abstraction of a proton. In the first case 5-aminolaevulinic acid would retain both methylene protons of glycine, in the second, one of the protons would be lost to the medium (Fig. 37). Acylation of the pyridoxal-bound intermediate (68 or 69) by succinyl-CoA would constitute the next step and this could be followed either by direct hydrolysis of the Schiff-base or by decarboxylation with subsequent hydrolysis depending on which course was chosen in the first stage of the reaction. 

The interest in the mechanisms of SchifF base hydrolysis stems largely from the fact that the formation and decomposition of SchifF base linkages play an important role in a variety of enzymatic reactions, for example, carbonyl transfers involving pyridoxal phosphate, aldol condensations, /3-decarboxylations and transaminations. The mechanisms for the formation and hydrolysis of biologically important SchifF bases, and imine intermediates, have been discussed by Bruice and Benkovic (1966) and by Jencks (1969). As the consequence of a number of studies (Jencks, 1959 Cordes and Jencks, 1962, 1963 Reeves, 1962 Koehler et al., 1964), the mechanisms for the hydrolysis of comparatively simple SchifF bases are reasonably well understood. From the results of a comprehensive kinetic investigation, the mechanisms for the hydrolysis of m- and p-substituted benzylidine-l,l-dimethylethylamines in the entire pH range (see, for example, the open circles in Fig. 13) have been discussed in terms of equations (23-26) (Cordes and Jencks, 1963) ... 

Kynureninase Kynureninase is a pyridoxal phosphate (vitamin Be) -dependent enzyme that catalyzes the hydrolysis of 3-hydroxykynurenine to... 

As with the normal mechanism of the enzyme, the inactivation starts with Schiff base formation with the enzyme-bound pyridoxal phosphate, followed by removal of an a-proton by an active-site base to form the reactive electrophilic intermediate (82). This then partitions between hydrolysis of the Schiff base linkage, resulting in the keto product (83)and enzyme reactivation, and Michael-type addition of an enzyme active-site nucleophile, resulting in a stable covalently bonded enzyme adduct (84). 

Hydrolysis of the imine then yields an a-keto acid along with a nitrogen-containing pyridoxal phosphate derivative. 

This reaction requires pyridoxal phosphate or pyridoxamine phosphate as a coenzyme (Scheme 8.8). If the former is used, it produces a Schiff base or enamine with the a-amino acid. The enamine undergoes an azaallylic transformation to form the alternative enamine. Hydrolysis of this produces pyridoxamine phosphate and the a-keto acid corresponding to the first amino acid. The pyridoxamine phosphate now forms an enamine with the first a-keto acid. Another azaallylic transformation takes place and the reaction is completed. It will be seen that there is no loss of ammonia or conversion of it into urea via the urea cycle. The transamination route simply shuffles the pack. 

Scission of the side chain leaves an Ai ion which takes up a pioton to give indole from tryptophan (as with tryptophanase) or phenol from tyrosine (as with 3-tyrosinase). The side chain of the original molecule is left as the pyridoxal phosphate complex of aminoacrylic acid, and on hydrolysis the aminoacrylic acid tautomerizes to the imine of pyruvic acid which is hydrolyzed to pyruvic acid and ammonia ... 

The pyridoxal phosphate complex of aminoacrylic acid can also be formed from serine by loss of an OH radical in a manner analogous to loss of the. 4.r" radical depicted above. This complex contains a reactive double bond to which the reactive /3-hydrogen of indole can add, giving a complex which on hydrolysis yields tryptophan. Such a mechanism is in accord with the known facts on tryptophan biosynthesis (cf. 858, and previous discussion, p. 41). 

The reaction mechanism consists of formation of a Schiff base by pyridoxal phosphate with a reactive amino group of the enzyme entry of glycine and formation of an enzyme-pyridoxal phosphate-glycine-Schiff base complex loss of a proton from the a carbon of glycine with the generation of a carbanion condensation of the carbanion with succinyl-CoA to yield an enzyme-bound intermediate (a-amino-yS-ketoadipic acid) decarboxylation of this intermediate to ALA and liberation of the bound ALA by hydrolysis. ALA synthesis does not occur in mature erythrocytes. 

The donor amino acid forms a Schiff base with pyridoxal phosphate within the enzyme s active site. After a proton is lost, a carbanion forms and is resonance-stabilized by interconversion to a quinonoid intermediate. After an enzyme-catalyzed proton transfer and a hydrolysis, the a-keto product is released. A second a-keto acid then enters the active site. This acceptor a-keto acid is converted to an a-amino acid product as the mechanism just described is reversed. 

Rosenthaler et al. [106] purified histidine decarboxylase from Lactobacillus 30A and demonstrated that there was no pyridoxal phosphate, as had been suggested by Rodwell [107]. Treatment with [ C]phenylhydrazine labeled the protein, but did not if the protein was first reduced with borohydride. Chymotrypsin digestion of the [ C]phenylhydrazone treated enzyme resulted in a labeled fragment identified as A -pyruvoylphenylalanine [100]. Borohydride reduction of the native enzyme resulted in lactate production after hydrolysis. Thus it was established that a pyruvoyl group is covalently bound as an amide to the NH 2-terminal phenylalanine. As is consistent with this proposed mechanism the enzyme is also inhibited by cyanide and by hydroxylamine. The iminium ion predicted by the mechanism above was trapped with borohydride in the presence of substrate and identified [108]. 

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