Properties of the Enzyme

The purpose of studies on enzyme properties is to select favorable reaction conditions for the investigation of enzyme kinetics. The choice of assay conditions has to be performed very carefully, and it has to be proven that the assay conditions are as close as possible to the reactor conditions of the final process. This aspect cannot be stressed too much  

The influence of all relevant reaction parameters on enzyme activity, selectivity and stability has to be considered. Parameters determining the enzyme properties are 

Measurement and modeling of the influence of concentrations of substrates, cosubstrates, coenzymes and inhibitors on enzyme activity form the central subject of enzyme kinetics and are discussed in Sect. 7.4. 

First of all the pH value and the temperature of the assay have to be chosen. The pH-optimum of the enzyme is determined by measuring the effect of pH on activity. It has to be recognized that the location of the pH-optimum depends on 

In addition the temperature dependency of enzyme activity must be measured, also yielding an optimum curve. This temperature optimum depends on the assay conditions, especially the incubation time, and is not, on its own, useful to identify a reasonable reaction temperature. Instead of this, the temperature stability of the enzyme has to be determined. To that end the enzyme is incubated with all relevant reaction components in test tubes, changing the temperature while keeping all other parameters constant. The assay conditions have to be as close as possible to the conditions relevant in the final process. In particular, stability measurements have to be performed in the presence of a relevant concentration of all substrates and coenzymes which have a stabilizing influence on the enzyme. 

We would now like to describe some of the properties of the enzyme and the requirements of the reaction. We want first to emphasize that the data are of a preliminary nature. Because of the extreme lability of the enzyme and the rapid loss of activity immediately after collection of the fractions, it is impossible at present to carry out quantitative experiments or study the kinetics of the reaction. The data given is therefore of a qualitative nature and not conclusive. 

The complete reaction mixt u e (70 nl) contained Tris-hydrochloride (pH 8), 50mM MgClp, 4njM KCl, lOOmM ATP, CTP, UTP, 2mM each (5h)-GTP, 0.06nmol (l5 Ci/mmol) actinomycin D, 5 EMC ENA, 

Recently, a new SAM dependent lysine 2,3-aminomutase was detected and characterized in Bacillus subtilis. Unlike the enzyme from C. subterminah SB4, the enzyme in B. subtilis apparently consists of four identical subunits each with a molecular mass of 54 kDa [30]. A PLP binding motif was identified in this amino-mutase that is also highly conserved in other lysine 2,3-aminomutases [31]. 

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Determination of DNA Sequence Information. Almost all DNA sequence is determined by enzymatic methods (12) which exploit the properties of the enzyme DNA polymerase. Whereas a chemical method for DNA sequencing exists, its use has been supplanted for the most part in the initial deterrnination of a sequence. Chemical or Maxam-Gilbett sequencing (13) is mote often used for mapping functional sites on DNA fragments of known sequence. 

It is worth noting that the reaction catalyzed by PEP carboxykinase (Eigure 20.25) could also function as an anaplerotic reaction, were it not for the particular properties of the enzyme. COg binds weakly to PEP carboxykinase, whereas oxaloacetate binds very tightly K, = 2 X 10 Af), and, as a result, the enzyme favors formation of PEP from oxaloacetate. 

Component B is a monomeric reductase with a molecular weight of 35,000 and contains per mol of enzyme, 1 mol of FMN, 2.1 mol of Fe, and 1.7 mol of labile sulfur. After reduction with NADH, the ESR spectrum showed signals that were attributed to a [2Fe-2S] structure and a flavo-semiquinone radical (Schweizer et al. 1987). The molecular and kinetic properties of the enzyme are broadly similar to the Class IB reductases of benzoate 1,2-dioxygenase and 4-methoxybenzoate monooxygenase-O-demethylase. 

Pepsin consists of a single polypeptide chain of molecular weight 34 644 and 327 amino acid residues. Ser-68 is phosphorylated, but this phosphate may be removed without significantly altering the catalytic properties of the enzyme. As in other acid proteases, the active site is an extended area that can accommodate... 

The difference in the effect of various endo-D-galacturonanases on oligomeric substrates [in particular, the difference in the rate of degradation of tri(D-galactosiduronic acid) and in the action pattern toward the tetrasaccharide] indicates, however, that it is not the substrate, but rather, the properties of the enzyme (in particular, the character of its active center) that constitute the determining factor. 

The hydrogenase may be electrically wired with different semiconductors, metals and conducting materials as an electrode. This property of the enzyme is successfully used in design of different biomolecular device for renewable energy production and conversion systems based on molecular hydrogen as intermediate energy carrier. In many cases... 

Even nowadays the application of radioactive isotopes is the most sensitive method for the analysis of biomolecules or their reaction products. Besides the low detection limits, the replacement of a naturally overbalancing stable isotope by its radioactive analogue does not interfere with the physical or chemical properties of the enzyme (with some exceptions for hydrogens). Figure 6 lists some frequently used radioactive isotopes and their half-life periods. 

Determination of the thermodynamic and kinetic parameters of interest requires monitoring of the surface concentration of the binding molecule. With large biomolecules, the surface concentrations are small, and simple redox labeling will not allow sufficient sensitivity. Labeling of the target biomolecule with a redox enzyme obviates this difficulty, thanks to the catalytic properties of the enzyme. 

With these possible applications in mind, we should review the significant characteristics of hydrogenase as a catalyst. Compared with most chemical catalysts, hydrogenases are large molecules. The protein has been selected by evolution, from an almost infinite number of possible structures. The whole protein is part of the machinery. Therefore even minor tampering with the protein, for example by site-directed mutagenesis, is likely to lead to unexpected changes in the properties of the enzyme. 

Stephenson, M. and Stickland, L. H. (1931) XXVII Hydrogenase a bacterial enzyme capable of activating molecular hydrogen. I. The properties of the enzyme. Biochem. J. (London), 25, 205-14. 

The use of D-AAO from the yeast Rhodotorula gracilis to deracemize naphthyl amino acids has been studied in some detail by the groups of Servi and Pollegioni, who compared the kinetic properties of the enzyme with racemic 1- and 2-naphthylalanine (1 and 2) and 1- and 2-naphthylglycine (3 and 4). 

Uyeda K, Rabinowitz JC. 1971. Pyruvate-ferredoxin oxidoreductase III. Purification and properties of the enzyme. J Biol Chem 246 311-19. 

The lowered temperature approach has been linked to flow, temperature jump, photolysis, and nmr methods. Cryoenzymology allows the characterization of enzyme intermediates which have life-times of only milliseconds at normal temperatures, but are stable for hours at low temperatures. Mixed aqueous/organic solvents or even concentrated salt solutions are employed and must always be tested for any adverse effects on the catalytic or structural properties of the enzyme. 

The other constant in the equation, is often used to compare enzymes. is the substrate concentration required to produce half the maximum velocity. Under certain conditions, is a measure of the affinity of the enzyme for its substrate. When comparing two eu2ymes, the one with the higher has a lower affinity for its substrate. The value is an intrinsic property of the enzyme-substrate system and cannot be altered by changing [S] or [E]. 

Pearce R, Greenway D, Parkinson A (1992) Species differences and interindividual variahon in liver microsomal cytochrome P450 2A enzymes effects on coumarin, dicumarol, and testosterone oxidation. Arch Biochem Biophys 298 211-225 Pelkonen O, Sotaniemi EA, Ahokas JT (1985) Coumarin 7-hydroxylase activity in human hver microsomes. Properties of the enzyme and interspedes comparisons. Br J Clin Pharmacol 19 59-66... 

The approach that is used to identify a regulatory mechanism in any biochemical process is discussed in Chapter 3. In brief, the approach is (i) identify the regulatory enzymes or processes (ii) study the properties of the enzymes or processes (iii) on the basis of the properties, formulate a theory of regulation (iv) test the theory. 

The enzyme catalyzing the addition of ADP-ribose units onto the histones and itself is poly(ADP-ribose) polymerase or synthetase. Poly(ADP-ribose) polymerase is a nuclear, DNA-dependent enzyme that is stimulated by DNA breaks [302]. This property of the enzyme would target its action to sites that have DNA strand breaks (regions of the genome involved in replication, repair, recombination). The enzyme is associated with chromatin areas and perichromatin regions in interphase Chinese hamster ovary cells [312]. Degradation of the ADP-ribose polymer is catalyzed by the nuclear enzyme poly(ADP-ribose) glycohydrolase and ADP-ribosyl protein lyase. 

These properties of the enzyme are good for the production of D-mannose from P-mannan in the presence of the P-mannanase described above. 

The reaction is exactly analogous to the chemical aldol reaction (also shown), but it utilizes an enamine as the nucleophile, and it can thus be achieved under typical enzymic conditions, i.e. around neutrality and at room temperature. There is one subtle difference though, in that the enzyme produces an enamine from a primary amine. We have indicated that enamine formation is a property of secondary amines, whereas primary amines react with aldehydes and ketones to form imines (see Section 7.7.1). Thus, a further property of the enzyme is to help stabilize the enamine tautomer relative to the imine. 

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