Pyridoxal-5 -phosphate synthase

Raschle T, Arigoni D, Brunisholz R, Rechsteiner H, Amrhein N, Fitzpatrick TB. Reaction mechanism of pyridoxal 5 -phosphate synthase. Detection of an enzyme-bound chromophoric intermediate. J. Biol. Chem. 2007 282 6098-6105. 

Moccand, C., Kaufmann, M., and Fitzpatrick, T.B. (2011) It takes two to tango defining an essential second active site in pyridoxal 5 -phosphate synthase. PLoS One, 6 (1), el6042. 

Hydrolases represent a significant class of therapeutic enzymes [Enzyme Commission (EC) 3.1—3.11] (14) (Table 1). Another group of enzymes with pharmacological uses has budt-ia cofactors, eg, in the form of pyridoxal phosphate, flavin nucleotides, or zinc (15). The synthases, and other multisubstrate enzymes that require high energy phosphates, are seldom available for use as dmgs because the required co-substrates are either absent from the extracellular space or are present ia prohibitively low coaceatratioas. 

Write a reasonable mechanism for the 3-ketosphinganine synthase reaction, remembering that it is a pyridoxal phosphate-dependent reaction. 

At the beginning of the MEP pathway, the glycolytic products, pyruvate and D-glyceraldehyde (GAP), are condensed in a transketolase reaction to deoxy-xylulose phosphate (DXP) by the deoxy-xylulose phosphate synthase (DXS) enzyme. DXP is the precursor for other pathways leading to pyridoxal and thiamine. 

Homocystinuria can be treated in some cases by the administration of pyridoxine (vitamin Bs), which is a cofactor for the cystathionine synthase reaction. Some patients respond to the administration of pharmacological doses of pyridoxine (25-100 mg daily) with a reduction of plasma homocysteine and methionine. Pyridoxine responsiveness appears to be hereditary, with sibs tending to show a concordant pattern and a milder clinical syndrome. Pyridoxine sensitivity can be documented by enzyme assay in skin fibroblasts. The precise biochemical mechanism of the pyridoxine effect is not well understood but it may not reflect a mutation resulting in diminished affinity of the enzyme for cofactor, because even high concentrations of pyridoxal phosphate do not restore mutant enzyme activity to a control level. 

As indicated in Section 6.3.3 and Table 6.2 the key control step is mediated by glycogen phosphorylase, a homodimeric enzyme which requires vitamin B6 (pyridoxal phosphate) for maximum activity, and like glycogen synthase (Section 6.2) is subject to both allosteric modulation and covalent modification. 

This pyridoxal-phosphate-dependent enzyme [EC 4.2.1.22] (also known as serine sulfhydrase, /3-thionase, and methylcysteine synthase) catalyzes the reaction of homocysteine with serine to produce cystathionine and water. 

This pyridoxal-phosphate-dependent enzyme [EC 4.2.99.9], also known as cystathionine y-synthase, catalyzes the reaction of O-succinyl-L-homoserine with L-cysteine to produce cystathionine and succinate. The enzyme can also use hydrogen sulfide and methanethiol as substrates, producing homocysteine and methionine, respectively. In the absence of a thiol, the enzyme can also catalyze a /3,y-elimination reaction to form 2-oxobu-tanoate, succinate, and ammonia. 

Gonversion of homocysteine to Gys occurs in two reactions catalyzed by two pyridoxal phosphate-requiring enzymes, cystathionine p-synthase and y-cystathionase. 

Among the numerous enzymes that utilize pyridoxal phosphate (PLP) as cofactor, the amino acid racemases, amino acid decarboxylases (e.g., aromatic amino acids, ornithine, glutamic acid), aminotransferases (y-aminobutyrate transaminase), and a-oxamine synthases, have been the main targets in the search for fluorinated mechanism-based inhibitors. Pharmaceutical companies have played a very active role in this promising research (control of the metabolism of amino acids and neuroamines is very important at the physiological level). 

The unusual amino acid, 1-aminocyclopropanecarboxylic acid, a precursor of the phytohormone ethylene, is biosynthesized in plants from S-adenosylmethionine. By using dideuterated S-adenosylmethionine, the reaction, under the influence of a pyridoxal phosphate dependent synthase, involves an inversion at the x-carbon center (a feature rarely observed for pyridoxal phosphate reactions), leading to (5)-l-amino-2,2-dideuterocyclopropanecarboxylic acid4. 

Formation of S-aminolevulinic acid (ALA) All the carbon and nitrogen atoms of the porphyrin molecule are provided by two simple building blocks glycine (a nonessential amino acid) and succinyl CoA (an intermediate in the citric acid cycle). Glycine and succinyl CoA condense to form ALA in a reaction catalyzed by ALA synthase (Figure 21.3) This reaction requires pyridoxal phosphate as a coenzyme, and is the rate-controlling step in hepatic porphyrin biosynthesis. 

Correct answer = B. The activity of 6-aminole-vulinic acid synthase controls the rate of por phyrin synthesis. The enzyme is increased in patients treated with certain drugs, and requires pyridoxal phosphate as a coenzyme. Another enzyme in the pathway ( -aminolevulinic acid dehydrase) is extremely sensitive to the pres ence of heavy metals. 

Committed step in heme synthesis, its coenzyme, and inhibitor The committed step in heme synthesis is the formation of 5-amlnolevulinic acid (ALA). The reaction, which requires pyridoxal phosphate as a coenzyme, is catalyzed by ALA synthase. The reaction is inhibited by hemin (the oxidized form of heme that accumulates in the cell when it is being under-used). The conversion of protoporphyrin IX to heme, catalyzed by ferrochelatase, is inhibited by lead. 

The vitamin biotin is formed in nature (left) by condensation of L-alanine with pimeloyl-CoAto form 8-amino-7-oxononanoate (AON). This compound is seen at the upper left of the center structure joined as a Schiff base with the coenzyme pyridoxal phosphate (PLP). This is a product complex of the enzyme AON synthase (see Webster et ah, Biochemistry 39,516-528,2000) Courtesy of D. Alexeev,... 

Figure 25-3 The structure of the two-enzyme a2p2 complex tryptophan synthase.65 66 The view is with the twofold axis of the OC2P2 complex vertical with the two a subunits at the ends and the P subunits in the center. The tunnel through which indole molecules released from indole propanol phosphate (IPP) in the a subunits to the pyridoxal phosphate (PLP) in the p subunits is shaded. Courtesy of C. Craig Hyde and Edith Wilson Miles.

Fig. 1. Ethylene biosynthesis. The numbered enzymes are (1) methionine adenosyltransferase, (2) ACC (l-aminocyclopropane-l-carboxylic acid) synthase, (3) ethylene forming enzyme (EFE), (4) 5 -methylthio-adenosine nucleosidase, (5) 5 -methylthioribose kinase. Regulation of the synthesis of ACC synthase and EFE are important steps in the control of ethylene production. ACC synthase requires pyridoxal phosphate and is inhibited by aminoethoxy vinyl glycine EFE requires 02 and is inhibited under anaerobic conditions. Synthesis of both ACC synthase and EFE is stimulated during ripening, senescence, abscission, following mechanical wounding, and treatment with auxins.

This reaction is catalyzed by the enzyme ALA synthase (Fig. 2d) which requires the coenzyme pyridoxal phosphate (see Topic M2) and is located in the mitochondria of eukaryotes. This committed step in the pathway is subject to regulation. The synthesis of ALA synthase is feedback-inhibited by heme. 

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

There are two pyridoxal phosphate-requiring enzymes in the homocysteine degradation pathway, which are associated with genetic diseases. In homo-cystinuria, cystathionine synthase is defective, and large amounts of homocystine are excreted in the urine. Some homocystinurics respond to the administration of large doses of vitamin B6. In cystathioninuria, cystathionase is either defective or absent. These patients excrete cystathionine in the urine. Cystathionase is often underactive in the newborns with immature livers, and cysteine and cystine become essential amino acids. Human milk protein is especially rich in cysteine, presumably to prepare the newborn for such a contingency. 

The transsulfuration pathway involves conversion of homocysteine to cysteine by the sequential action of two pyridoxal phosphate (vitamin B6)-dependent enzymes, cystathionine- 5-synthase (CBS) and cystathionine y-lyase (Fig. 21-2). Transsulfuration of homocysteine occurs predominantly in the liver, kidney, and gastrointestinal tract. Deficiency of CBS, first described by Carson and Neill in 1962, is inherited in an autosomal recessive pattern. It causes homocystinuria accompanied by severe elevations in blood homocysteine (>100 (iM) and methionine (>60 (iM). Homocystinuria due to deficiency of CBS occurs at a frequency of about 1 in 300,000 worldwide but is more common in some populations such as Ireland, where the frequency is 1 in 65,000. Clinical features include blood clots, heart disease, skeletal deformities, mental retardation, abnormalities of the ocular lens, and fatty infiltration of the fiver. Several different genetic defects in the CBS gene have been found to account for loss of CBS activity. 

Figure 21-1. Structural and metabolic relationships between methionine, homocysteine, and cysteine. CBS, cystathionine b-synthase CTH, cystathionine y-lyase MAT, methionine adenosyltransferase MS, methionine synthase 5-MTHF, 5-methyltetrahydrofoIate MTs, methyl transferases PLR pyridoxal phosphate SAH, S-adenosylhomocysteine SAHH, SAH hydrolase THF, tetrahydrofolate.

The first step of porphyrin synthesis is the condensation of succinyl-CoA and glycine to form 8-aminolevulinate. The reaction takes place in mitochondria, where succinyl-CoA is available. The reaction is irreversible and requires pyridoxal phosphate and Mg2+. It is catalyzed by the enzyme 8-aminolevulinate synthase. 

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. 

Jhee KH, McPhie P, and Miles EW (2000) Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein. Journal of Biological Chemistry 275,11541. ... 

Kabil O, Toaka S, LoBrutto R, Shoemaker R, and Banerjee R (2001) Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases. Evidence from P NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme. Journal of Biological Chemistry 276,19350-5. 

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