Enzymes substrate range

Kinetic studies involving enzymes can principally be classified into steady and transient state kinetics. In tlie former, tlie enzyme concentration is much lower tlian that of tlie substrate in tlie latter much higher enzyme concentration is used to allow detection of reaction intennediates. In steady state kinetics, the high efficiency of enzymes as a catalyst implies that very low concentrations are adequate to enable reactions to proceed at measurable rates (i.e., reaction times of a few seconds or more). Typical enzyme concentrations are in the range of 10 M to 10 ], while substrate concentrations usually exceed lO M. Consequently, tlie concentrations of enzyme-substrate intermediates are low witli respect to tlie total substrate (reactant) concentrations, even when tlie enzyme is fully saturated. The reaction is considered to be in a steady state after a very short induction period, which greatly simplifies the rate laws. 

Until 1987, the (R)-PaHNL from almonds was the only HNL used as catalyst in the enantioselective preparation of cyanohydrins. Therefore, it was of great interest to get access to HNLs which catalyze the formation of (5 )-cyanohydrins. (5 )-SbHNL [EC 4.1.2.11], isolated from Sorghum bicolor, was the first HNL used for the preparation of (5 )-cyanohydrins. Since the substrate range of SbHNL is limited to aromatic and heteroaromatic aldehydes as substrates, other enzymes with (5 )-cyanoglycosides have been investigated as catalysts for the synthesis of (5 )-cyanohydrins. The (5 )-HNLs from cassava (Manihot esculenta, MeHNL) and from Hevea brasiliensis (HbHNL) proved to be highly promising candidates for the preparation of (5 )-cyanohydrins. Both MeHNL and HbHNL have been overexpressed successfully in Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. 

Maximal speed (Vmax) and supposed Michaelis constant (K ) of pectin hydrolysis reaction (catalyzed by the studied pectinesterase) were determined in Zinewedwer — Berk coordinated, They were determined in the range of substrate concentration values that was below optimum one V = 14.7 10 M min K = 5.56 10 M. The value of dissociated constant (KJ of the triple enzyme—substrate complex was determined from the experimental data at high substrate concentration. It was the following Kj= 0.22 M. Bunting and Murphy method was used for determination. 

The taxonomic application of the ability of enteric organisms to grow with 4-hydroxy-phenylacetate (Cooper and Skinner 1980) and 3-hydroxyphenylpropionic acid (Burlingame and Chapman 1983) has been established. In addition, it has been demonstrated that the enzyme that carries out the hydroxylation has a wide substrate range extending to 4-meth-ylphenol, and even to 4-chlorophenol (Prieto and Garcis 1994). 

Methane monooxygenase may exist in either soluble (sMMO) or particulate (pMMO) forms. These display different substrate ranges and different rates of transformation rates, and most methanotrophs express only the latter form of the enzyme (Hanson and Hanson 1996). The particulate form of methane monooxygenase contains copper, or both copper... 

Enzyme activity generally passes through a maximum as the pH of the system in question is varied. However, the optimum pH varies with substrate concentration and temperature. Provided that the pH is not changed too far from the optimum value corresponding to the maximum rate, the changes of rate with pH are reversible and reproducible. However, if the solutions are made too acid or too alkaline, the activity of the enzyme may be irreversibly destroyed. Irreversible deactivation is usually attributed to denaturation of the proteinaceous enzyme. The range of pH in which reversible behavior is observed is generally small and this... 

Interestingly, a significant level of alkyl BTs was also removed. This suggests that the substrate range of the dsz enzymes from R. erythropolis includes a significant range of alkyl BTs. The report also indicated that significant level of sulfidic molecules were also removed, but that their identity could not be deciphered due to the presence of other hydrocarbons. 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

Potentiometric enzyme-based electrodes have found application in clinical, pharmaceutical, food and biochemical analyses to enable the selective determination of a wide range of important enzyme substrates, including amino acids, esters, amides, acylcholines, /Mactam antibiotics, sugars, enantioselective drugs and many others [74]. 

These CYPs are isoenzymes (or isozymes), catalysing essentially the same reaction, but for different substrate ranges with specificity determined by their different amino acid sequences. CYPs (and other metabolic enzymes) often react with individual substrates in a highly regio-, chemo- or stereo-selective manner, each isozyme displaying its own unique selectivity. Some examples of selective CYP-catalysed transformations are shown in Scheme 1.3. 

We have found a new (/ )-hydroxynitrile lyase from Japanese apricot (P. mume). The new enzyme accepts a broad array of substrates, ranging from aromatic, heteroaromatic, bicyclic to aliphatic carbonyl compounds, and yields the corresponding cyanohydrins with excellent enantioselection. 

If an approximate Km value for the enzyme-substrate combination of interest is known, a full-scale kinetic assay may be done immediately. However, often an approximate value is not known and it is necessary first to do a range finding or suck and see preliminary assay. For such an assay, a concentrated substrate solution is prepared and tenfold serial dilutions of the substrate are made so that a range of substrate concentrations is available within which the experimenter is confident the Km value lies. Initial velocities are determined at each substrate concentration, and data may he plotted either hyperholically (as V versus [S]) or with [S] values expressed as logio values. In the latter case, a sigmoidal curve is fitted to data with a three parameter logistic equation (O Eq. 4) ... 

Work in solution is an absolute prerequisite for further studies of enzyme-substrate intermediates in the crystalline state. According to the Arrhenius relationship, k = A exp(—E IRT), which relates the rate constant k to the temperature, reactions normally occurring in the second to minute ranges might be sufficiently decreased in rate at subzero temperatures to permit intermediates to be stabilized, and occasionally purified by column chromatography if reactions are carried out in fluid solvent mixtures. Therefore, the first problem is to find a suitable cryoprotective solvent for the protein in question. 

Tables VIII-XI show examples of pon variations of several buffers. With such tables, it is easy to adjust any desired pan value in mixed solvents at any selected temperature or in a given range of temperatures. We will see in Section III,E how these values are essential to investigate safely both crystal structure and productive enzyme-substrate complexes in the crystalline state.

The hbraries of enzyme substrates were obtained by spht-pool synthesis to yield one-bead-one-compound hbraries. The substrate assay was performed with a range of proteolytic enzymes such as subtilisin Carlsberg [26], cruzipain [27], protein disulfide isomerase [28-29], matrix metalloprotease MM P-9 [30], papain [31],... 

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