Enzyme cascade

Calcium is essential to several steps in the enzyme cascade of the blood clotting process, such as the conversion of prothrombin to thrombin (23). Clotting can be inhibited in stored blood suppHes by addition of complexing agents such as EDTA or citrate which reduce the levels of the free ion, Ca(Il). 

C9. Carson, S. D and Johnson, D. R., Consecutive enzyme cascades Complement activation at the cell surface triggers increased tissue factor activity. Bloodl6,361-367 (1990). 

Another interesting case is the one-pot four-enzyme cascade conversion of glycerol into a heptose sugar on gram scale [11], in which a pH switch method is applied to temporarily turn off one of the enzymes involved (Fig. 13.6). The four consecutive enzymatic conversion steps in one and the same reactor, without separation of intermediates, consist of ... 

An impressive one-pot six-step enzymatic synthesis of riboflavine from glucose on the laboratory scale has been reported with an overall yield of 35-50%. Six different enzymes are involved in the various synthesis steps, while two other enzymes take care for the in situ cofactor regenerations [12]. This example again shows that many more multi-enzyme cascade conversions will be developed in the near future, as a much greater variety of enzymes in sufficient amounts for organic synthetic purposes will become available through rapid developments in genomics and proteomics. 

One of the established methods for finding hits is high-throughput screening (HTS). This is a powerful method enabling several thousands of compounds per day to be tested. However, there are drawbacks. In many cases the assay for the detection of a hit comprises coupled enzymatic reactions, and a test compound may falsely lead to positive results because of its interference with components of the enzyme cascade. Problems may arise further from the chemical substances that make up the compound depository which are tested in HTS. Especially for companies with historically grown collections of compounds, the quality of the chemicals is an uncontrolled parameter in the sense that it is not always known whether the compounds are still unmodified they may have decomposed, precipitated or formed aggregates (polymers). 

A different and equally important way of achieving a rapid and significant change of flux is the amplification inherent in enzyme cascades. Here, each enzyme uses as its substrate the next enzyme in the sequence. In even a simple three-step cascade, the amplification achieved can be significant. For example assuming each step has an... 

Nitric oxide and eicosanoid synthesis haem synthesis. The importance of the pentose phosphate pathway reduced glutathione in maintaining red cell integrity. The respiratory burst in phagocytes. Clotting and complement enzyme cascades. Metabolism of lipoproteins. 

The protein-based clotting process is a classic example of an enzyme cascade (see Figure 5.23). The clotting factors (which are designated with a Roman numeral, I to XIII) are synthesized in the liver and circulate in the blood as inactive precursors, strictiy, proenzymes. Most of the clotting factors are serine protease enzymes, that is they are enzymes which cleave other proteins (substrates) by a mechanism which involves a serine residue at the active site. 

N. P. Structure of an E6AP-UbcH7 complex insights into ubiquitination by the E2-E3 enzyme cascade. Science 1999, 286, 1321-26. 

Once autophosphorylation begins, a complex of other events ensues. An insulin receptor substrate (IRS-1) binds the receptor and is phosphorylated on tyrosine residues, allowing proteins with SH2 (src homology) domains to bind to the. phosphotyrosine residues on IRS-1 and become active. In this way, the receptor activates several enzyme cascades, which involve ... 

Among antihypertensives, those targeting the renin-angiotensin system are among the best studied. The renin-angiotensin system consists of a two-enzyme cascade that is involved in the regulation of blood pressure and electrolyte homeostasis. 

The cause of this disease is not known, but one factor that increases blood pressure is a plasma protein, angiotensin-II, which is produced via an enzyme cascade in blood, as follows ... 

This is described as a cellular cascade since it has some similarity to an enzyme cascade (see Chapter 3) that is, a single B-cell produces a very large number of effector cells which produce a very large number of antibodies (Figure 17.9). 

As in blood coagulation (see p. 290), the early components in the complement system are serine proteinoses, which mutually activate each other through limited proteolysis. They create a self-reinforcing enzyme cascade. Factor C3, the products of which are involved in several functions, is central to the complement system. 

The diverse effects of insulin (see p. 160) are mediated by protein kinases that mutually activate each other in the form of enzyme cascades. At the end of this chain there are kinases that influence gene transcription in the nucleus by phosphorylating target proteins, or promote the uptake of glucose and its conversion into glycogen. The signal transduction pathways involved have not yet been fully explained. They are presented here in a simplified form. 

At the center of the apoptotic process lies a group of specialized cysteine-containing aspartate proteinases (see p. 176), known as cas-pases. These mutually activate one another, creating an enzyme cascade resembling the cascade involved in blood coagulation (see... 

The classical example is blood clotting, where successive steps involving enzyme-catalyzed proteolysis converts an inactive (or weakly active) proenzyme into its highly active form. Although unknown at the time of Wald s classical report, kinase-type and nucleotidyltransferase-type reactions (See Enzyme Cascade Kinetics) are frequently the source of biological signal transduction and amplification. 

A relationship used in the analysis of enzyme cascade systems. It is symbolized by S (and also referred to as the sensitivity index) and is equal to 8.89[eo,5]/([eo,9] [eo.i]) where [60,5] is the concentration of effector required to attain 50% maximal amplitude, [eog] is the concentration of effector required for 90% maximal am-phtude, [eo.i] is the concentration of effector required for 10% maximal amphtude, and 8.89 is a normalizing constant used to yield a reference value of 1.00 for a pure hyperbolic binding isotherm ... 

A parameter used to assist in the characterization of enzyme cascade systems. Symbolized by S A, it is equal to [eo.5E]/[eo.5i] where [eo.sE] is the concentration of effector required for 0.5 activation of the converter enzyme E and [eo.5i] is the concentration of effector at which 50% of the interconvertible enzyme (1) has been modified See Enzyme Cascade Kinetics P. B. Chock E. R. Stadtman (1980) Meth. Enzymol. 64, 297. 

ENZYME CASCADE KINETICS AMPLITUDE AMP NUOLEOSIDASE AMYLASES... 

ENZYME CASCADE KINETICS BLUE COPPER PROTEIN Blue shift,... 

AFFINITY LABELING ENZYME CASCADE KINETICS COX-YEATS EQUATION C—P bond biosynthesis,... 

ENZYME CASCADE KINETICS Enzyme catalytic model,... 

RIBULOSE-5-PHOSPHATE 3-EPIM ERASE Escherichia coll glutamine synthetase, ENZYME CASCADE KINETICS GLUTAMINE SYNTHETASE Essential amino acid residues in catalysis, AFFINITY LABELING ESTERASES... 

ISOTOPE EXCHANGE AT EQUILIBRIUM WEDLER-BOYER TECHNIQUE CUMULATIVE INHIBITION UNCONSUMED SUBSTRATE CRYPTIC CATALYSIS BOROHYDRIDE REDUCTION ENZYME CASCADE KINETICS... 

In the multi-enzyme system described by Sheldon et al. [15], the key point is the use of the phosphatase phytase from Aspergillus ficuum, which is a cheap and readily available industrial enzyme. Phytase is active at acid pH and becomes inactive at neutral pH. Thus, the pH can be used to switch on/off the activities of the various enzymes, allowing us to carry out the four-enzyme cascade in one pot. 

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