Alternative substrates catalyzed

Isoflavones have been implicated in goiter induction. Soybean extracts inhibit reactions catalyzed by thyroid peroxidase (TPO), essential to the synthesis of thyroid hormones (Divi et al., 1997). Genistein and daidzein (at about 1-10 p,M of IC50) may act as alternative substrates for tyrosine iodination (Divi et al., 1997). Furthermore, genistein and daidzein have also been shown to cause the irreversible inactivation of TPO in the presence of hydrogen peroxide. Genistein also inhibits thyroxine synthesis in the presence of iodinated... 

This enzyme [EC 1.3.1.43], also referred to as arogenate dehydrogenase and pretyrosine dehydrogenase, catalyzes the reaction of arogenate with NAD+ to produce tyrosine, NADH, and carbon dioxide. Both prephenate and D-prephenyllactate can act as alternative substrates. 

Regioselectivity can also have stereochemical issues associated with the catalytic event. For example, glutamine synthetase will catalyze amide bond formation using /3-glutamate as an alternative substrate to produce each of the stereoisomeric /3-glutamine products, albeit at different rates. 

This pyridoxal-phosphate-dependent enzyme [EC 4.4.1.16], also known as selenocysteine reductase, catalyzes the reaction of L-selenocysteine with a reduced acceptor to produce hydrogen selenide, L-alanine, and the acceptor. The enzyme can use dithiothreitol or 2-mercaptoethanol as the reducing agent. Cysteine, serine, or chloroalanine are not alternative substrates for this enzyme. 

These were differently affected by different procedures. For example, when the enzyme was activated at 55°, the increment in ki was slight, but k2 increased 3.5-fold. Similarly, in the presence of EDTA, fc, and k2 values decreased independently, suggesting that the sites for both activities were different. Center and Behai (5) found that with the P. mirabilis enzyme, cyclic 2, 3 -UMP competitively inhibited the hydrolysis of bis(p-nitrophenyl) phosphate. The Ki was 40 pAf very close to the Km for the cyclic nucleotide (Km, 75 yM) which indicated that the two compounds could serve as alternate substrates being hydrolyzed at the same active site. In contrast, 3 -AMP was a mixed inhibitor of cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate hydrolysis. Adenosine was a mixed inhibitor of bis(p-nitrophenyl) phosphate hydrolysis but a competitive inhibitor of 3 -AMP hydrolysis. From such kinetic studies Center and Behai (5) suggested that two separate and adjacent sites A and B are involved in the hydrolysis of the diester and phos-phomonoester substrates. Site A serves as a binding site for hydrolysis of ribonucleoside 2, 3 -cyclic phosphates and together with site B catalyzes the hydrolysis of the diester bond. During this reaction 3 -... 

The requirement of the phosphatase that dephosphorylates KD0-8-phosphate is very specific (Table IV). None of the phos-phorylated sugars tested was either an inhibitor or an alternate substrate for this phosphatase. The specificity of this enzyme, the fact that none of the other intracellular phosphatases catalyze the hydrolysis of KD0-8-phosphate and the fact that KD0-8-phosphate needs to be dephosphorylated for subsequent metabolism... 

Alternative substrates may exist for the PHDs proposed examples include RNA polymerase II and IkB kinase-P (which is negatively regulated by PHDl) (115, 116). However, unequivocal evidence (e.g., demonstration of hydroxylation by mass spectrometry) has not yet been demonstrated for these proteins. In contrast, FIH has been shown to catalyze hydroxylation of ankyrin repeat domain (ARD) proteins from the NFkB (nuclear factor kB) and Notch family at highly conserved as-paraginyl residues (117, 118). The ARD is a common protein motif, with over 200 human members of the ARD protein family being predicted. Evidence that ARD hydroxylation occurs frequently in human cells supports the assertion (117, 118) that posttranslational hydroxylation of cytoplasmic proteins in... 

An alternate approach, which also uses enzyme-catalyzed ring-opening of a lactone to generate a mechanism-activated inhibitor, was developed by Katzenellenbogen and his co-workers [183], who found enol lactones, exemplified by (13-8) and (13-9), to be potent, selective inhibitors of HLE. The haloenol lactone (13-9) was an irreversible inactivator of HLE and chymotrypsin, and after exposure to (13-9), active enzyme could not be regenerated even upon treatment with hydrazine. Enol lactone (13-8), on the other hand, was an alternate-substrate inhibitor, which produced only transient inhibition of HLE and chymotrypsin. These results have been interpreted to mean that, with the halo-substituted compounds, ring opening results in formation of an acyl-enzyme that contains a reactive halomethyl ketone, which then alkylates His-57. That these compounds... 

The inhibition of the hexokinase-catalyzed reaction between glucose and ATP by fructose or mannose is an example of competitive inhibition by alternate substrates. Glucose, fructose, and mannose are all substrates of hexokinase and can be converted to product (hexose-6-phosphate). All three hexoses combine with the enzyme at the same active site. Consequently, the utilization of any one of the hexoses is inhibited in the presence of either of the other two. The reaction scheme describing dead-end competitive inhibition is ... 

Two pinene cyclases have been isolated from sage (19,35). Electrophoretically pure pinene cyclase I converts geranyl pyrophosphate to (+)-a-pinene and to lesser quantities of (+)-camphene and (+)-limonene, whereas pinene cyclase II, of lower molecular weight, converts the acyclic precursor to (-)-B-pinene and to lesser quantities of (-)-a-pinene, (-)-camphene and (-)-limonene. Both purified enzymes also utilize neryl and linalyl pyrophosphate as alternate substrates for olefin synthesis. The availability of enzyme systems catalyzing formation of enantiomeric products from a common, achiral substrate has provided an unusual opportunity to examine the stereochemistry of cyclization. 

Second, the enzyme also catalyzes the oxygenation of ribulose bisphosphate, a reaction in which O2 serves as an alternate substrate and is competitive against CO2 [Eq. (15)]. 

Scheme 39 Reaction catalyzed by LgtC using (a) the natural substrate UDP a-D-galactose or (b) the alternative substrate 2,4-dinitrophenyl /3-D-galactoside.

SHMT also catalyzes racemization of alanine and transamination of both its enantiomers. The particular reaction catalyzed by SHMT is mainly determined by the structure of the amino acid substrate. In the case of serine or glycine, the true substrates, SHMT does not catalyze any of the alternate reactions. The currently accepted model attributes this reaction specificity to the existence of open and closed active-site conformations. The physiological substrates generate the closed conformation, whereas alternate substrates react while the enzyme remains in the open conformation, which permits reaction paths leading to decarboxylation, transamination, and racemization. ... 

Comparisons of the kinetic coefficients in Eq. (1) obtained from initial rate measurements with alternative substrates have given a considerable amount of information about reaction pathways as well as indications of the molecular basis of specificity (60). This approach, much used for proteolytic enzymes, has been exploited particularly with the alcohol dehydrogenases, which catalyze the oxidation of a variety of primary and secondary alcohols (61). While several other dehydrogenases have been studied in this way, most of the results have been reported only as apparent maximum rates and apparent Km values for the alternative substrate, which restricts the amount of information that can be derived. 

This category refers to enzymes that catalyze different reactions (and not just different substrates) than the one they evolved for. As is the case with substrate and coenzyme ambiguity, the enzyme s activity with these alternative substrates is purely accidental, and is under no selection, and is therefore promiscuous by definition. As suggested, these cases include chemical transformations where the bonds that are broken, or formed, are different than those in the native substrate and reaction, and/or transformations that proceed through a different transition state. As discussed later, the promiscuous chemical transformations can be performed by the same catalytic side chains, and by essentially the same mechanism, as the native enzymatic function (Section 8.03.6). But there are also cases in which the enzyme utilizes different subsets of active-site residues, and somewhat different mechanisms, for the native and promiscuous functions (Section 8.03.6.1.4). 

Extracted FeMoco is not active for the reduction of N2. However, it has been observed to reduce protons to H2 electrochemically. It also reacts with alternative substrates (Section 8.22.3.3) for example, it binds multiple molecules of cyanide. It will catalyze the reduction of acetylene to ethylene and ethane by metal amalgam reducing agents. In an interesting observation, N2 is a competitive inhibitor of acetylene reduction, providing the first evidence for N2 binding at extracted FeMoco. ... 

The presence of the second enzyme in the pathway in higher plants can be inferred from results obtained by Saytanrayana and Radhakrishnan (1965). NADPH-dependent conversion of the acetohydroxyacids to the oxo-analogues of isoleucine and valine indicated that partially purified extracts of Phaseolis radiatus contained the enzymes required to catalyze both the second and third reactions illustrated in Fig. 4. Reductoisomerase activity would require NADPH for synthesis of the dihydroxyacids which would, in turn, be dehydrated for synthesis of the oxoacids. The rates of NADPH oxidation differed when 2-acetolactate and 2-acetohydroxybutyrate were tested as alternate substrates, but it was not established whether more than one reductoisomerase was present in the preparations. 

Figure 4.52 Reactions catalyzed by the taxoid lOp-hydroxylase, taxoid 13a-hydroxylase, and taxoid 14p-hydroxylase. AU of these hydroxylases accept either the 5a-alcohol or the corresponding acetate ester as alternate substrates. Only the most favorable routes are illustrated.

Alternatively, enzyme-catalyzed reactions may be performed in nonconven-tional media composed of microemulsions and liquid crystals [135]. The use of these systems, however, requires a great deal of knowledge of bioprocess engineering for the separation of the surfactant from substrate(s) and/or product(s). 

Differentiation between reaction mechanisms can be achieved by careful scmtiny of the K versus substrate concentration patterns (Fig. 7.4). The adage that a picture tells a thousand words is quite applicable in this instance. It is difficult to determine the mechanism of an enzyme-catalyzed reaction from steady-state kinetic analysis. The determination of the mechanism of an enzymatic reaction is neither a trivial task nor an easy task. The use of dead-end inhibitors and alternative substrates, study of the patterns of product inhibition, and isotope-exchange experiments... 

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