Phosphate as inhibitor

Shinohara K and Sakurai H (198O) 3-Naphthyl oligophosphate. Inhibitors of photophosphorylation and H+-ATPase of spinach chloroplasts. Plant Cell Physiol 21, T5 8i+ (1981) Pyridoxal 5 phosphate, phenyl phosphate and acetyl phosphate as inhibitors of photophosphorylation competitive with phosphate. Plant Cell Physiol. 22, li 7 1 5T (1982) Light-induced ATP formation from acetyl... 

PHOSPHOFRUCTOKINASE shows positive cooperativity with fructose-e-phosphate as the substrate. ATP, an allosteric inhibitor, binds to the T state and decreases the velocity. AMP, a signal for low energy, binds to the R state and increases the velocity of the reaction. 

Aryl phosphites are widely used as inhibitors of oxidation. Phosphites react with hydroperoxides and are oxidized into phosphates by the following stoichiometric reaction [1-10] ... 

Scheme 18.37 3/i-Substituted steroids as inhibitors of glucose-6-phosphate dehydrogenase. 

In addition to the aforementioned allenic steroids, prostaglandins, amino acids and nucleoside analogs, a number of other functionalized allenes have been employed (albeit with limited success) in enzyme inhibition (Scheme 18.56) [154-159]. Thus, the 7-vinylidenecephalosporin 164 and related allenes did not show the expected activity as inhibitors of human leukocyte elastase, but a weak inhibition of porcine pancreas elastase [156], Similarly disappointing were the immunosuppressive activity of the allenic mycophenolic acid derivative 165 [157] and the inhibition of 12-lipoxygenase by the carboxylic acid 166 [158]. In contrast, the carboxyallenyl phosphate 167 turned out to be a potent inhibitor of phosphoenolpyruvate carboxylase and pyruvate kinase [159]. Hydrolysis of this allenic phosphate probably leads to 2-oxobut-3-enoate, which then undergoes an irreversible Michael addition with suitable nucleophilic side chains of the enzyme. 

Only compounds of type I and II, which contain both metal and sulfur, were found to act as inhibitors. Compounds III and IV, for example, were ineffective. The inhibition of indene oxidation by zinc dithio-phosphates was considered to be a key result in this work since it rules out the intermediate formation of free thiyl radicals. There is adequate evidence (2, 9, 16, 17, 22) that thiyl radicals, including (RO)2PS2, add rapidly to olefins and that in the presence of oxygen the following sequence of reactions would occur. 

Canaline is a potent inhibitor of all seven pyridoxal phosphate-containing enzymes studied by Rahiala et (27) but it lacks adverse effects on three ornithine-utiTTzing enzymes lacking a Bg cofactor. Finally, in jack bean, Canavalia ensiformis, ornithine carbamoyl transferase can form 0-ureido-L-homoserine from canaline and carbamoyl phosphate as it does citrulline from ornithine and carbamoyl phosphate. Nevertheless, neither compound inhibited formation of the reaction products (31). 

Lindell and Turner have subsequently applied the methodology to the synthesis of diflu-oromethylene phosphonates for evaluation as inhibitors of aspartate transcarbamoylase (equation 110)152. Similarly, Chambers and coworkers utilized this method in the preparation of 2-amino-l,l-difluoroethylphosphonic acid, a phosphate mimic (equation 111)153. 

The X-ray crystal structure of the inorganic phosphate (an inhibitor) complex of alkaline phosphatase from E. coli (9) showed that the active center consists of a Zn2Mg(or Zn) assembly, where the two zinc(II) atoms are 3.94 A apart and bridged by the bidentate phosphate (which suggests a phosphomonoester substrate potentially interacting with two zinc(II), as depicted in Fig. 2), and the Mg (or the third Zn) is linked to one atom of the zinc pair by an aspartate residue at a distance... 

Although casein is a substrate for milk acid phosphatase, the major caseins, in the order as(asl + as2) > (3 > k, also act as competitive inhibitors of the enzyme when assayed on p-nitrophenylphosphate, probably due to binding of the enzyme to the casein phosphate groups (the effectiveness of the caseins as inhibitors is related to their phosphate content). 

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 fairly complicated effects of varied pH on the differential patterns of participation of various phosphate compounds as substrates and as inhibitors, as described in detail above, also may be rationalized on the basis of this mechanism. Prevailing ionic species of some phosphate substrates at various pH values are pictured in Fig. 8, structures (X)-(XIX), along with relevant pXa values. Glucose-6-P2- (XVII), PPi2 (X), nucleosidediphosphate2 (XII), nucleosidetriphosphate3- (XIV), mannose-6-P2- (see XVII), and carbamyl-P2- (XIX) are effective both as substrates and as inhibitors, for both phenomena are very prominent... 

The carbohydrate moiety of RNA is D-ribose with the / -D-ribofurano-side ring. The 2 - and 3 -OH groups are cis to each other and easily form the cyclic phosphate intermediate. Although the 2 -deoxynucleotides bind to the enzyme, they only serve as inhibitors. The 2 -OH group is mandatory for the catalytic activity of pancreatic RNase. 

Ribonuclease A hydrolyzes RNA adjacent to pyrimidine bases. The reaction proceeds through a 2, 3 -phosphate cyclic diester intermediate. Formation and breakdown of the cyclic diester appear to be promoted by concerted general-base and general-acid catalysis by two histidine residues, and by electrostatic interactions with two lysines. These reactions proceed through pentavalent phosphoryl intermediates. The geometry of these intermediates resembles the geometry of vanadate compounds that act as inhibitors of the enzyme. 

A full consideration of the mechanism of the sodium pump requires an account of the role of the lipid, the binding sites for Na+, K+, Mg2+ and ATP, the mechanism of hydrolysis of ATP and the way in which this is coupled to the transport of the cation. In addition it should be noted that the enzyme also functions as a K+-dependent phosphatase, a reaction usually studied with p-nitrophenyl phosphate as substrate. Studies with inhibitors have been informative, notably with ouabain and with vanadate. Ouabain binds at one site per pump and so has been of value in quantitatively defining the enzyme in various preparations. 

The phosphate derivatives (301)-(303) of 6-0-(2-hydroxyethyl)cyclohexane-l,2,4,6-tetraol have been synthesized as inositol monophosphatase inhibitors, the putative target for lithium therapy.270 Compounds (303) and (302) are the most potent examples of a primary alkyl phosphate and phosphate monanion inhibitor so far reported. 

The data in Table III indicate that the enzyme KD0-8-phos-phate synthase is more difficult to inhibit with phosphorylated substrate analogues than is D-arabinose-5-phosphate isomerase. Non-phosphorylated substrate analogues (Tables II) were also tested as inhibitors or substrates of KDO-8-phosphate synthase but as with D-arabinose-5-phosphate isomerase, these analogues were... 

---