Enzyme kinetic properties

L2. Lakomek, M Huppke, P Neubauer, B Pekrun, A., Winkler, H., and Schroter, W., Mutations in the R-type pyruvate kinase gene and altered enzyme kinetic properties in patients with hemolytic anemia due to pyruvate kinase deficiency. Ann. Hematol. 68,253-260 (1994). 

The optimum yield of a condensation product is obtained at the pH where Ka has a maximum. For peptide synthesis with serine proteases this coincides with the pH where the enzyme kinetic properties have their maxima. For the synthesis of penicillins with penicillin amidase, or esters with serine proteases or esterases, the pH of maximum product yield is much lower than the pH optimum of the enzymes. For penicillin amidase the pH stability is also markedly reduced at pH 4-5. Thus, in these cases, thermodynamically controlled processes for the synthesis of the condensation products are not favorable. When these enzymes are used as catalysts in thermodynamically controlled hydrolysis reactions an increase in pH increases the product yield. Penicilhn hydrolysis is generally carried out at pH about 8.0, where the enzyme has its optimum. At this pH the equiUbrium yield of hydrolysis product is about 97%. It could be further increased by increasing the pH. Due to the limited stability of the enzyme and the product 6-aminopenicillanic acid at pH>8, a higher pH is not used in the biotechnological process. 

Al. Abrahamson, M., Barrett, A. J., Salvesen, G., and Grubb, A., Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J. Biol. Chem. 261(24), 11282—11289 (1986). 

The /3-galactosidase study is an excellent example of the power of site-directed mutagenesis. Huber, Miller, and colleagues prepared and examined five Glu-461-/8-galactosidase substitutions (Asp, Gly, Gin, His, and Lys) (136, 139). All substitutions had /teat values less than 0.3% of the wild-type enzyme except the His-461 mutation, which was approximately 6%. For most of the substitutions it was possible to quantify K, /teat. s. and rates of galactosylation and degalac-tosylation for three substrates, and K values for three inhibitors. Different enzyme kinetic properties resulting from different amino acid substitutions confirm that Glu-461 is directly involved in catalysis and contributes to active site structure stability. Heat inactivation at 55°C occurred more rapidly with each amino acid substitution compared to the wild-type enzyme, except for the structurally conservative Gin substitution, which was only moderately affected. 

Enzymic activities of crude soil suspensions have been demonstrated to follow Michaelis-Menten kinetics. Calculated Km values have varied for different soils and for active fractions of soil extracts. The extent to which kinetic constants of soil enzymes are influenced by the state in which the enzymes occur in soils, is unknown. For some activities, two Km values have been distinguished for the one crude soil extract. Fractionation has revealed that enzymes may exist in tightly- and loosely- bound complexes with soil coloured humic compounds. Enzymes freed of coloured materials may nevertheless still be bound in complexes by their association with carbohydrates. These may not only influence enzyme kinetic properties but evidence suggests that they may also confer some degree of stability on the enzymes in soil. Whereas early speculation on the mechanism(s) by which enzymes are protected in soil tended, in the case of abiontic enzymes, to focus on the role of clay or humic colloids, fractionation studies have drawn attention to the potentially important role of soil carbohydrates. The manner in which carbohydrates are bonded to the enzymes in soil has not as yet been established and may be a fruitful line of enquiry. 

Measurements of particular properties of a compound or substance (enzyme kinetics, reaction kinetics, FACS, fluorescence-activat cell sorting, assay). 

The basic kinetic properties of this allosteric enzyme are clearly explained by combining Monod s theory and these structural results. The tetrameric enzyme exists in equilibrium between a catalytically active R state and an inactive T state. There is a difference in the tertiary structure of the subunits in these two states, which is closely linked to a difference in the quaternary structure of the molecule. The substrate F6P binds preferentially to the R state, thereby shifting the equilibrium to that state. Since the mechanism is concerted, binding of one F6P to the first subunit provides an additional three subunits in the R state, hence the cooperativity of F6P binding and catalysis. ATP binds to both states, so there is no shift in the equilibrium and hence there is no cooperativity of ATP binding. The inhibitor PEP preferentially binds to the effector binding site of molecules in the T state and as a result the equilibrium is shifted to the inactive state. By contrast the activator ADP preferentially binds to the effector site of molecules in the R state and as a result shifts the equilibrium to the R state with its four available, catalytically competent, active sites per molecule. 

Dixon, M., et al., 1979. Enzymes, 3rd ed. New York Academic Press. A classic work on enzyme kinetics and die properties of enzymes. 

The multiple human variants of G-6-PD and the relationship of their kinetic properties to the presence of hemolytic anemia under conditions which closely simulate the erythrocyte internal environment have been elegantly studied by Yoshida (112). This author found that those enzyme variants with significantly reduced physiological activity are those associated with hemolytic anemia. 

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. 

Hereditary deficiency of phosphoglycerate kinase (PGK) is associated with hereditary hemolytic anemia and often with central nervous system dysfunction and/or myopathy. The first case, reported by Kraus et al. (K24), is a heterozygous female, and the results are not so clear. The second family, reported by Valentine et al. (V3), is a large Chinese family, whose pedigree study indicates that PGK deficiency is compatible with X-linked inheritance. To date, 22 families have been reported (04, T25, Y3). Nine of these have manifested both symptoms five have shown only hemolysis seven have shown the central nervous system dysfunction and/or myopathy but without hemolysis and one case, PGK Munchen, is without clinical symptoms (F5). PGK II is an electrophoretic variant found in New Guinea populations (Y2). Red blood cell enzyme activity, specific activity, and the kinetic properties of this polymorphic variant are normal. 

In M. rhodesianum no isoenzymes of the 3-ketothiolase have been found [ 14]. The enzyme s capability to react with long-chain acyl-CoAs has not been tested. The enzyme was found to be very similar to the 3-ketothiolases from R. eutropha, A. beijerinckii, Z. ramigera, and R. ruber with respect to molecular weight, optimum pH, and kinetic properties [14]. 

The synthesis of 5-HT can increase markedly under conditions requiring more neurotransmitter. Plasticity is an important concept in neurobiology. In general, this refers to the ability of neuronal systems to conform to either short- or long-term demands placed upon their activity or function (see Plasticity in Ch. 53). One of the processes contributing to neuronal plasticity is the ability to increase the rate of neurotransmitter synthesis and release in response to increased neuronal activity. Serotonergic neurons have this capability the synthesis of 5-HT from tryptophan is increased in a frequency-dependent manner in response to electrical stimulation of serotonergic soma [7]. The increase in synthesis results from the enhanced conversion of tryptophan to 5-HTP and is dependent on extracellular calcium ion. It is likely that the increased 5-HT synthesis results in part from alterations in the kinetic properties of tryptophan hydroxylase, perhaps due to calcium-dependent phosphorylation of the enzyme by calmodulin-dependent protein kinase II or cAMP-dependent protein kinase (PKA see Ch. 23). 

Brain hexokinase is inhibited by its product glucose-6-phosphate and to a lesser extent by adenosine diphosphate. The isoenzyme of hexokinase found in brain may be soluble in the cytosol or be attached firmly to mitochondria [2 and references therein]. An equilibrium exists between the soluble and the bound enzyme. The binding changes the kinetic properties of hexokinase and its inhibition by Glc-6-P resulting in a more active enzyme. The extent of binding is inversely related to the ATP ADP ratio, i.e. conditions in which energy utilization... 

Manipulation of one enzymatic step in a system can have wide reaching consequences because of the interplay between metabolite levels and a wide range of regulatory circuits. These circuits can operate at the level of transcription, translation, post-translational modification, or through allosteric and competitive influences on the kinetic properties of enzymes. 

The next section describes the utilization of //PLC for different applications of interest in the pharmaceutical industry. The part discusses the instrumentation employed for these applications, followed by the results of detailed characterization studies. The next part focuses on particular applications, highlighting results from the high-throughput characterization of ADMET and physicochemical properties (e.g., solubility, purity, log P, drug release, etc.), separation-based assays (assay development and optimization, real-time enzyme kinetics, evaluation of substrate specificity, etc.), and sample preparation (e.g., high-throughput clean-up of complex samples prior to MS (FIA) analysis). 

As discussed earlier, the tacit assumption of in vitro studies is that they are faithful reporters of how the enzymes and substrates will behave in vivo. At least qualitatively, the assumption seems largely to be true but quantitatively the assumption is less reliable. It assumes that the different microenvironments surrounding an enzyme in vivo and in an in vitro preparation do not differentially affect kinetic properties. It also assumes that, given equal concentrations of drug, the concentration that actually reaches the active site of the enzyme in the two different microenvironments will be equal (5). Clearly this does not need to be the case. As a consequence, a more reliable reporter of the in vivo kinetic properties of a drug would be highly desirable. 

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