PBG synthase

The growth, porphyrin excretion, and the activity of the first three enzymes involved in the synthesis of tetrapyrrole skeleton from glycine and succinyl-CoA, ALA synthase, PBG synthase, and PBG deminase, were measured under excretion (ethanol/malate/glutamate, 40°C) and non-excretion (ethanol/NaHC03/NH4Cl, 40°C) conditions (Figures 2 and 3). 

Growth (OD660) was almost the same, but there was a great difference in A401 for the two conditions. ALA synthase activity tends to decrease in the time course, but is considerably higher in excretion condition than in non-excretion condition. PBG synthase activity, also higher in excretion condition, shows five fold increase compared to the start of incubation. PBG deaminase activity is maintained at constant level, but higher in excretion conditions. Relative activity of ALA synthase is approximately one tenth of those of PBG synthase and deaminase. This elevated level of biosynthetic enzymes seems to be one reason for enhanced porphyrin excretion. 

PBG synthase is a metal-requiring enzyme but the metals required vary from one source to another. The bovine enzyme requires zinc, the E. coli enzyme binds both zinc and magnesium [16,17], whereas plant enzymes only require magnesium. In the bovine enzyme two zinc atoms bind per subunit. An EXAFS experiment has shown that the first zinc ion, required for activity, binds in a site... 

Chlorolaevulinic acid 28 is a potent competitive inhibitor of bovine PBG synthase, presumably due to binding in the active site in place of ALA, and it also inactivates the enzyme by alkylation of a specific cysteine residue [24] (Scheme 9). The concentration required for the inactivation is much greater than that required for competitive inhibition, however, which suggests that the processes occur at different sites on the enzyme. Electrospray mass spectrometry has shown that 28 can alkylate at multiple sites on the B. subtilis enzyme without causing more than about 50% loss of activity [18]. It is likely that there is no cysteine residue in the active site of this latter enzyme. 

It will be obvious from the above that there is still considerable uncertainty about the mechanism of PBG synthase. However there have been reports of crystallisation of the bovine [26] and yeast enzymes [27], and preliminary X-ray... 

Lead inhibits PBG synthase and ferrochelatase, restricting haem biosynthesis and resulting in microcytic hypochromic anaemia and porphyria. Urinary excretion of 5-ALA is increased. 

PBG synthase (EC 4.2.1.24) acts on two equivalents of 5-aminolevulinate to produce the condensation product, prophobilinogen (PBG) [5-(aminomethyl)-4-(carboxy-methyl)-lH-pyrrole-3-propanoic acid] and two equivalents of water (Scheme 14.26). The pathway has been examined in some detail and it appears that imines are formed with two separate lysine residues. The imines undergo condensation. ... 

A summary of the steps in the biosynthesis of the porphyrin derivatives from PBG is given in Figure 32-8. The last three enzymes in the pathway and ALA synthase are located in the mitochondrion, whereas the other enzymes are cytosolic. Both erythroid and non-erythroid ( housekeeping ) forms of the first four enzymes are found. Heme biosynthesis occurs in most mammalian cells with the exception of mature erythrocytes, which do not contain mitochondria. However,... 

Figure 32-6. Conversion of porphobilinogen to uroporphyrinogens. Uroporphyrinogen synthase I is also called porphobilinogen (PBG) deaminase or hydroxy-methylbilane (HMB) synthase.

If the patient is actually asymptomatic, but has a family history of acute porphyria or prior symptoms suspicious of acute porphyria, hydroxymethylbilane synthase (HMBS) activity, plasma scanning, and fecal porphyrins should be measured. These tests will reveal AIP, P V, and HC. As a small percentage of AIP families exhibit normal HMBS activity, PBG in a urine sample can be added. PBG determination can also performed as a first choice, if an acute porphyria is suspected. But if normal, it does not exclude acute porphyrias in asymptomatic phases. Furthermore, the existence of an acute porphyria is only proved if the value exceeds at least five times the upper limit of normal. 

The stereochemistry of the final deprotonation, at the carbon which becomes C-2 of PBG,has been determined using [5S- H] ALA (derived from [2- 111glycine by the ALA synthase reaction, see Sect. 2.2) [1,2]. The tritium label is entirely retained in the PBG produced, whereas 50% is lost from ALA which is randomly tritiated at C-5. Thus it is the pro-J hydrogen atom that is lost as illustrated in Scheme 8,27 16. 

Figure 2-1 Schematic representation of the heme biosynthetic pathway in mammalian cells. ALAS, S-aminolevulinate synthase PBGS, porphobiUnogen synthase PBGD, porphobilinogen deaminase Uro III synthase, uroporphyrinogen III synthase Uro III decarboxylase, uroporphyrinogen III decarboxylase CPO, coproporphyrinogen oxidase PPO, protoporphyrinogen oxidase FC, ferrochelatase.

Fig 1 Separation of a soluble extract from the cyanobacterium Synechococcus 6301 on Sephacryl S-300. The UV-pattem, measured at 280 nm and the activities for PBG-deaminase, ALA-dehydratase, GSA-aminotransferase and glutamyl-tRNAGlu-Synthase are given. 

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