Enzyme Multiplicity

Like the P450 multigene family, there are several different isozymes present in two gene families. There are three major gene families  

FIGURE 3.4 Structures of endogenous compounds involved in glucuronidation bile adds, lithocholic add (LA) and hyodeoxycholic acid (HDCA) short chain bile acids, etianic add and isoetianic add, steroid hormones, androsterone, testosterone, estrone, estradiol, estriol. 

UGTl—various forms catalyze conjugation of planar phenols, bulky phenols, amines, tertiary amines, and bilirubin. (Nine active human forms now cloned are expressed, i.e., lAl, 1A3-1A10). 

UGT2B—xenobiotics, steroids and bile acids ( 4 human active enzymes, i.e., 2B4, 2B7, 2B10, 2B15, 2B17). 

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McKenna NJ, Xu J, Nawasz Z, Tsai SY, O Malley BW (1999) Nuclear receptor coactivators multiple enzymes, multiple complexes, multiple functions. J Steroid Biochem Mol Biol 69 3-12... 

SHIELDING MULTI ENZYME Multiple attack enzyme,... 

Sophisticated regulation can also evolve by duplication of the genes encoding the biosynthetic enzymes. For example, the phosphorylation of aspartate is the committed step in the biosynthesis of threonine, methionine, and lysine. Three distinct aspartokinases catalyze this reaction in E. coli, an example of a regulatory mechanism called enzyme multiplicity. (Figure 24.24). The catalytic domains of these enzymes show approximately 30% sequence identity. Although the mechanisms of catalysis are essentially identical, their activities are regulated differently one enzyme is not subject to feedback inhibition, another is inhibited by threonine, and the third is inhibited by lysine. 

All three must be inhibited before production of the intermediate ceases completely. In addition, each of the amino acids inhibits the first enzyme on its branch line away from aspartate-semialdehyde, so ensuring that the decrease in concentration of the metabolite affects only the production of the inhibiting amino acid. This is an example of control by enzyme multiplicity, although the individual inhibitions are brought about by the type of allosteric processes that have already been described. 

The homogeneous assay is based on a competitive equilibrium between Ag and Ag for a limited amount of antibody as described (see Fig. 14 and Section 11.2). The assay relies on a decrease in the current signal for the reduction/oxidation of Ag when bound to Ab. Since all of these assays have no enzyme multiplication feature, the detection limits are generally much higher. In the model assay that follows, the free, equilibrium concentration of electroactively labeled estriol is determined by differential pulse polarography at a dropping mercury electrode (Wehmeyer et al., 1982). The peak current from the polarographic reduction of Ag is then proportional to the concentration of Ag present. The separation of Ab Ag from Ag is unnecessary because of the attenuation of the Ab Ag reduction. 

Define the committed step of a metabolic pathway and recognize that it is often the target of feedback regulation. Note the main features of control of branched pathways by feedback inhibition and activation, enzyme multiplicity, and cumulative feedback. 

Enzyme multiplicity The first common step is catalyzed by two or more isozymes, one is inhibited by E and the other by G E. coli 2-Keto-3-deoxy-D-arabinoheptonate synthetase one isozyme is inhibited by Phe and the other by Tyr... 

Chiral stationary phases that are currently available can be classified into those containing cavities (cellulose derivatives, cyclodextrins, synthetic polymers, crown ethers, and chiral imprinted gels), affinity phases (bovine serum albumin, human serum albumin, a-glycoprotein, enzymes), multiple hydrogen-bond phases, Ti-donor and Ti-acceptor phases, and chiral ligand exchange phases. This classification scheme was used in a review that gave numerous pharmaceutical examples of separation by... 

Edson KZ, Rettie AE (2013) CYP4 Enzymes as potential drug targets focus on enzyme multiplicity, inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid omega-hydroxylase activities. Curr Top Med Chem 13 1429-1440... 

A variety of patterns of end-product inhibition have been described [5,69] (1) In enzyme multiplicity inhibition balanced control of an early enzyme of the common part of a branched pathway is obtained because the enzyme is present in the form of several isoenzymes, each specifically inhibited by an end product of one of the branches. (2) In cumulative feedback inhibition an enzyme which mediates the formation of a product used in many pathways is partly inhibited by individual end products of the pathways. Each inhibitor adds its effect to the total inhibition, but the combined effect is less than the sum of the single inhibitions. (3) In concerted feedback inhibition two or more end products are required to act together before any significant inhibition is exhibited. (4) In cooperative feedback inhibition several end products can act as partial inhibitors of an enzyme, but a mixture of two different inhibitors results in greater inhibition than the sum of the individual inhibitions. (5) The term sequential feedback inhibition refers to inhibition of an early enzyme by an intermediate whose accumulation is controlled by inhibition of one or more late pathway enzymes by the end product [71 ]. 

The synthesis of tryptophan in microorganisms is affected at several levels by end-product inhibition. Thus, end-product feedback inhibition partly regulates the synthesis of chorismic acid which is the final product of the common aromatic pathway and serves as a substrate for the first reaction in the tryptophan-synthesizing branch pathway (see Fig. 2). Regulation of the common aromatic pathway was recently reviewed by Doy [72]. The first enzyme of the common aromatic pathway, 3-deoxy-D-flrah/>jo-heptulosonate 7-phosphate synthetase (DAHPS), has been reported to exist as at least three isoenzymes, each specifically susceptible to inhibition by one of the aromatic amino acid end products (tyrosine, phenylalanine, and tryptophan), in E. coli (see reference [3]). It should be noted that many reports have indicated that in E. coli the DAHPS (trp), the isoenzyme whose synthesis is repressed specifically by tryptophan, was not sensitive to end-product inhibition by tryptophan. Recently, however, tryptophan inhibition of DAHPS (trp) activity has been demonstrated in E. coli [3,73,74]. The E. coli pattern, therefore, represents an example of enzyme multiplicity inhibition based on the inhibition specificity of isoenzymes. It is interesting to note the report by Wallace and Pittard [75] that even in the presence of an excess of all three aromatic amino acids enough chorismate is synthesized to provide for the synthesis of the aromatic vitamins whose individual pathways branch from this last common aromatic intermediate. In S. typhimurium, thus far, only two DAHPS isoenzymes, DAHPS (tyr) and DAHPS (phe) have been identified as sensitive to tyrosine and phenylalanine, respectively [76]. 

Many difficulties have to be faced during the purification of tRNA methyltransferases. The major ones are ii) enzyme multiplicity, which makes it cumbersome to isolate each specific reaction product, and then difficult to evaluate specific activity and extent of purification (ii) enzyme instability (Hi) frequent contamination by ribonuclease activity (iv) possible presence of complexes with endogenous tRNA (v) unavailability of a proper tRNA substrate, as we have already discussed. To point out the problem of... 

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