Phosphates enzymic reactions

Two or more linked enzyme reactions can lead to a change in the concentration of NADH or NADPH that is equivalent to the concentration of the original analyte. The reference glucose measurement using hexokinase [9001-51-8] and glucose-6-phosphate dehydrogenase [9001-40-5] is an example ... 

FIGURE 19.18 A mechanism for the glycer-aldehyde-3-phosphate dehydrogenase reaction. Reaction of an enzyme snlfliydryl with the carbonyl carbon of glyceraldehyde-3-P forms a thiohemiacetal, which loses a hydride to NAD to become a thloester. Phosphorolysls of this thloester releases 1,3-blsphosphoglycerate. 

Interestingly, however, the mechanisms of the two phosphate hydrolysis reactions in steps 9 and 11 are not the same. In step 9, water is the nucleophile, but in the glucose 6-phosphate reaction of step 11, a histidine residue on the enzyme attacks phosphorus, giving a phosphoryl enzyme intermediate that subsequently reacts with water. 

Enzyme Assay. Na , K -ATPase, and sarcoplasmic reticulum Ca - ATPase were prepared from rat hearts (22) and dog hearts (23), respectively. Bovine heart cyclic AMP phosphodiesterase was purchased from Sigma. The enzyme reaction was carried out after 5-min pretreatment with the drug, and the amount of inorganic phosphate liberated during the reaction period was determined. 

Ribulose 5-phosphate is capable of a reversible isomerization to other pentose phosphates-xylulose 5-phosphate and ribose 5-phosphate. These reactions are catalyzed by two respective enzymes, viz., pentose-phosphate epimerase and pentose-phosphate isomerase, according to the scheme below ... 

Subsequent studies196 on crystalline transketolase have revealed the presence of a contaminating enzyme termed pentulose 5-phosphate waldenase (or epimerase) the presence of which had led to the erroneous conclusion that d-erythro-pentulose 5-phosphate was the substrate for transketolase. d-erythro-Pentulose 5-phosphate is virtually unattacked by transketolase prepared from spinach or liver. In subsequent discussions of experiments involving the use of transketolase, in this article, the enzymic reactions must be viewed as the result of action of transketolase and pentulose 5-phosphate waldenase (epimerase). 

The reaction catalyzed by KDO 8-phosphate synthetase (reaction 2, Scheme 35) was first observed by Levin and Racker9 in extracts from Pseudomonas aeruginosa (see Scheme 1), and later by Ghalambor and Heath29 in extracts from Escherichia coli 0111 B4 and J-5. In the initial experiments of Levin and Racker,135 the fate of D-ribose 5-phosphate in crude bacterial extracts was studied, and the KDO 8-phosphate discovered by the authors is really derived from D-ribose 5-phosphate by three, sequential, enzyme-catalyzed reactions (see Scheme 36). 

These circumstances became apparent to the authors when they attempted to study the formation of KDO 8-phosphate as catalyzed by purified bacterial extracts. These extracts did not catalyze the formation of KDO 8-phosphate from D-ribose 5-phosphate, but required D-arabinose 5-phosphate as the substrate Heath and Ghalambor29 showed that the KDO 8-phosphate synthetase reaction, observed in Pseudomonas extracts by Levin and Racker, is also catalyzed by extracts from Escherichia coli strains 0 111 B4 and J-5. Rick and Osborn136 showed that the KDO 8-phosphate synthetase from a Salmonella typhimurium mutant conditionally defective in cell-wall synthesis had a KM of 6 mM as compared to a KM of 170 pM for the enzyme from wild-type cells. 

Many methods have been developed in which a product of the reaction is chemically modified to produce a substance with a particular spectral property. The inorganic phosphate released by the hydrolysis of phosphate esters may be measured by simple chemical methods (Fiske and Subbarow) after the enzyme reaction has been stopped. Such techniques are often convenient but do not lend themselves to the measurement of initial velocity. 

A few years later, in 1953, the versatility of pyridoxal phosphate was illustrated by Snell and his collaborators who found many of the enzyme reactions in which pyridoxal phosphate is a coenzyme could be catalyzed non-enzymically if the substrates were gently heated with pyridoxal phosphate (or free pydridoxal) in the presence of di- or tri-valent metal ions, including Cu2+, Fe3+, and Al3+. Most transaminases however are not metal proteins and a rather different complex is formed in the presence of the apoprotein. 

A different application of visible microscopy was pioneered by Gomori. In 1941 he showed that alkaline phosphatase could be specifically located by its hydrolysis of soluble phosphate esters (initially glycerophosphate). If calcium ions were present in the medium in which the sections were incubated, insoluble calcium phosphate precipitated as a result of the action of the hydrolase. The site of the precipitate could be visualized if cobalt or lead salts were subsequently added to replace calcium and the sections exposed to hydrogen sulfide. In principle many hydrolases and other enzymes could be studied using the appropriate substrates and precipitants. It was important to ensure that the products of the enzyme reactions did not diffuse from the sites where the enzymes were located. It was also essential that the reagents could reach the enzyme site. 

Analyses of enzyme reaction rates continued to support the formulations of Henri and Michaelis-Menten and the idea of an enzyme-substrate complex, although the kinetics would still be consistent with adsorption catalysis. Direct evidence for the participation of the enzyme in the catalyzed reaction came from a number of approaches. From the 1930s analysis of the mode of inhibition of thiol enzymes—especially glyceraldehyde-phosphate dehydrogenase—by iodoacetate and heavy metals established that cysteinyl groups within the enzyme were essential for its catalytic function. The mechanism by which the SH group participated in the reaction was finally shown when sufficient quantities of purified G-3-PDH became available (Chapter 4). 

Figure 4.21 Monitoring of an enzyme reaction using size-exclusion liquid chromatography. Column, TSK GEL G3000SW, 60 cm x 7.5 mm i.d. eluent, 0.07 M potassium phosphate buffer containing 0.1 M potassium chloride flow rate, 1 ml min-1 detection, UV 280 nm. Peaks 1, fl-lactoglobulin 2, a-chymotrypsin, and 3, decomposed products.

Table 11.6 Observed and intrinsic kinetic isotope effects on the glucose-6-phosphate dehydrogenase reaction in D2O (Cleland, W. W. in Cook, P. F., Ed., Enzyme Mechanism from Isotope Effects CRC Press, Boca Raton FL, 1991. Hermes, J. D. and Cleland, W. W. J. Am. Chem. Soc. 106, 7263 (1999))...

The Schiff base can undergo a variety of reactions in addition to transamination, shown in Fig. 6.4 for example, racemization of the amino acid via the a-deprotonated intermediate. Many of these reactions are catalyzed by metal ions and each has its equivalent nonmetallic enzyme reaction, each enzyme containing pyridoxal phosphate as a coenzyme. Many ideas of the mechanism of the action of these enzymes are based on the behavior of the model metal complexes. 

Pyridoxal phosphate is a required coenzyme for many enzyme-catalyzed reactions. Most of these reactions are associated with the metabolism of amino acids, including the decarboxylation reactions involved in the synthesis of the neurotransmitters dopamine and serotonin. In addition, pyridoxal phosphate is required for a key step in the synthesis of porphyrins, including the heme group that is an essential player in the transport of molecular oxygen by hemoglobin. Finally, pyridoxal phosphate-dependent reactions link amino acid metabolism to the citric acid cycle (chapter 16). 

The terminology vitamin Bg covers a number of structurally related compounds, including pyridoxal and pyridoxamine and their 5 -phosphates. Pyridoxal 5 -phosphate (PLP), in particular, acts as a coenzyme for a large number of important enzymic reactions, especially those involved in amino acid metabolism. We shall meet some of these in more detail later, e.g. transamination (see Section 15.6) and amino acid decarboxylation (see Section 15.7), but it is worth noting at this point that the biological role of PLP is absolutely dependent upon imine formation and hydrolysis. Vitamin Bg deficiency may lead to anaemia, weakness, eye, mouth, and nose lesions, and neurological changes. 

In both cases, the mixed anhydride is used to synthesize ATP from ADP. Hydrolysis of the anhydride liberates more energy than the hydrolysis of ATP to ADP and, therefore, can be linked to the enzymic synthesis of ATP from ADP. This may be shown mechanistically as a hydroxyl group on ADP acting as nucleophile towards the mixed anhydride, and in each case a new phosphoric anhydride is formed. In the case of succinyl phosphate, it turns out that GDP rather than ADP attacks the acyl phosphate, and ATP production is a later step (see Section 15.3). These are enzymic reactions therefore, the reaction and the nature of the product are closely controlled. We need not concern ourselves why attack should be on the P=0 rather than on the C=0. 

Most people have heard of antihistamines, even if they have little concept of the nature of histamine. Histamine is the decarboxylation product from histidine, and is formed from the amino acid by the action of the enzyme histidine decarboxylase. The mechanism of this pyridoxal phosphate-dependent reaction will be studied in more detail later (see Section 15.7). 

The first step is carboxylation of acetyl CoA to malonyl CoA. This reaction is catalyzed by acetyl-CoA carboxylase [5], which is the key enzyme in fatty acid biosynthesis. Synthesis into fatty acids is carried out by fatty acid synthase [6]. This multifunctional enzyme (see p. 168) starts with one molecule of ace-tyl-CoA and elongates it by adding malonyl groups in seven reaction cycles until palmi-tate is reached. One CO2 molecule is released in each reaction cycle. The fatty acid therefore grows by two carbon units each time. NADPH+H is used as the reducing agent and is derived either from the pentose phosphate pathway (see p. 152) or from isocitrate dehydrogenase and malic enzyme reactions. 

This enzyme [EC 4.2.99.8], also known as cysteine synthase and O-acetylserine sulfhydrylase, catalyzes the pyr-idoxal-phosphate-dependent reaction of H2S with O -acetylserine to produce cysteine and acetate. Some alkyl thiols, cyanide, pyrazole, and some other heterocyclic compounds can also act as acceptors. 

This enzyme [EC 2.6.1.2], also known as glutamic-pyruvic transaminase and glutamic-alanine transaminase, catalyzes the pyridoxal-phosphate-dependent reaction of alanine with 2-ketoglutarate, resulting on the production of pyruvate and glutamate. 2-Aminobutanoate will also react, albeit slowly. There is another alanine aminotransferase [EC 2.6.1.12], better known as alanine-oxo-acid aminotransferase, which catalyzes the pyridoxal-phosphate-dependent reaction of alanine and a 2-keto acid to generate pyruvate and an amino acid. See also Alanine Glyoxylate Aminotransferase... 

This enzyme [EC 2.6.1.21], also known as D-aspartate aminotransferase, D-amino acid aminotransferase, and D-amino acid transaminase, catalyzes the reversible pyridoxal-phosphate-dependent reaction of D-alanine with a-ketoglutarate to yield pyruvate and D-glutamate. The enzyme will also utilize as substrates the D-stereoisomers of leucine, aspartate, glutamate, aminobutyrate, norva-hne, and asparagine. See o-Amino Acid Aminotransferase... 

This enzyme [EC 2.6.1.18], also known as j8-alanine-pyruvate aminotransferase, catalyzes the reversible pyridoxal-phosphate-dependent reaction of /3-alanine with pyruvate to generate 3-oxopropanoate and alanine. 

This enzyme [EC 2.6.1.44] catalyzes the reversible, pyri-doxal-phosphate-dependent reaction of alanine with gly-oxylate to generate pyruvate and glycine. One component of the animal enzyme can utilize 2-oxobutanoate as a substrate instead of glyoxylate. A second component of the enzyme can also catalyze the reaction of alanine with 3-hydroxypyruvate to produce pyruvate and serine. See SerineiPyruvate Aminotransferase... 

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