Kinetic studies alkaline phosphatase

Deming and Pardue studied the kinetics for the hydrolysis of p-nitrophenyl phosphate by the enzyme alkaline phosphatase. The progress of the reaction was monitored by measuring the absorbance due to p-nitrophenol, which is one of the products of the reaction. A plot of the rate of the reaction (with units of pmol mL s ) versus the volume, V, (in milliliters) of a serum calibration standard containing the enzyme yielded a straight line with the following equation... 

Because ATP and GTP are required in many biosynthetic and mechanochemical reactions, the ability to manipulate their concentrations is particularly important in many kinetic studies. The three enzymes most commonly employed for this purpose are hexokinase, phosphofruc-tokinase, and alkaline phosphatase. Each of these enzymes offers advantages and disadvantages, and one must consider the design requirements for any particular experiment before using one of these to deplete ATP or GTP. 

Neither the occurrence of a constant value of Vmax or a constant product ratio is sufficient proof of the presence of an intermediate. It was seen for alkaline phosphatase that a constant value for Vmax is an artifact, and also that there is no a priori reason why the attack of acceptors on a Michaelis complex should not also give constant product ratios. In order for partitioning experiments to provide a satisfactory proof of the presence of an intermediate, they must be linked with rate measurements. When the rate measurements are restricted to steady state kinetics, the most favorable situation is when the intermediate accumulates. If the kinetics of equations 7.5 to 7.7 hold, it may be concluded beyond a reasonable doubt that an intermediate occurs. The ideal situation is a combination of partitioning experiments with pre-steady state studies, as described for chymotrypsin and amides. 

Early kinetic studies on alkaline phosphatase-catalyzed hydrolyses were consistent with the following scheme 106, 123, 128, 145, 146) ... 

Another enzyme that was studied extensively in microreactors to determine kinetic parameters is the model enzyme alkaline phosphatase. Many reports have appeared that differ mainly on the types of enzyme immobilization, such as on glass [413], PDMS [393], beads [414] and in hydrogels [415]. Kerby et al. [414], for example, evaluated the difference between mass-transfer effects and reduced effidendes of the immobilized enzyme in a packed bead glass microreactor. In the absence of mass-transfer resistance, the Michaelis-Menten kinetic parameters were shown to be flow-independent and could be appropriately predicted using low substrate conversion data. 

Although a considerable literature records numerous studies in this field (A19, B17, C12, J3, L9, P16, S4, S5), many have been performed on preparations of tissues from other than human sources. In conformity with the subject of this chapter and to avoid species differences, most attention will be directed to human tissue alkaline phosphatases and in particular their variants. The stereospecific L-phenylalanine inhibition has provided the impetus to study its molecular mechanism, which necessarily requires an understanding of the mechanism of catalysis. It is expected that discovery of other stereospecific inhibitors will follow and that they may have even greater utility than L-phenylalanine. However, since it is the first such unique inhibitor, this section of the chapter will receive extensive treatment after a consideration of some basic kinetic information. 

The problem of elucidating the mechanism of catalysis by alkaline phosphatase has been attacked by a number of workers from different directions from kinetic studies and modification of key linkages in the substrates and enzyme to the visualization of mechanism through models. Studies of related phosphohydrolases are relevant (A19, K26, L20, Pll, P12). 

An interesting study on the fate of injected placental alkaline phosphatase (N17) has been reported by Posen et al. (P18), who measured heat-stable alkaline phosphatase. Following injection, there was an initial rapid fall in enzyme activity lasting 3-5 hours and then a less rapid disappearance. After 4 hours, two thirds of the initial activity remained, but it took several weeks for complete disappearance. These kinetics resembled those known for serum albumin labeled with iodine-131. 

A16. Anagnostopoulos, C., and Matsudaira, H., Purification and kinetic studies of the alkaline phosphatase of human placenta. Proc. Intern. Symp. Enzyme Chtm., Tokyo Kyoto, 1957 (K. Ichihara, ed.), p. 166. Manizen, Tokyo, 1958. 

A phosphoryl-enzyme intermediate is formed during reactions catalysed by alkaline phosphatase. 0-4-Nitrophenyl phosphorothioate is hydrolysed 1000 times more slowly by alkaline phosphatase than is its oxygen analogue, suggesting an 5n2(P) mechanism for the phosphorylation of the enzyme. From a kinetic study of the reaction between a series of alkyl-, aryl-, and arylamido-phosphates and alkaline phosphatase, it has been shown that steric factors are important in these reactions. Moreover, although amidophosphates [e.g. (69)] are substrates for this enzyme ... 

Regarding bacteria, the purified alkaline phosphatase (EC 3.1.3.1) associated with the outer membrane of Lysobacter enzymogenes UASM 495 (ATCC 29487) was most active at pH 8.5 and two values (0.056 and 0.34 mM) were estimated from kinetic studies using para-nitrophenyl phosphate (von Tigerstrom and Stelmas-chuk, 1986). 

Zhang, J. and A. E. G. Cass, 2006. Kinetic study of site directed and randomly immobilized his-tag alkaline phosphatase by flow injection chemiluminescence. /. Mol. Recognit. 19 243-246. 

Ludikhuyze, L, Claeys, W and Hendrickx, M, (2000) Combined pressure-temperature inactivation of alkaline phosphatase in bovine milk a kinetic study. Journal of Food Science, 65, 155-160,... 

A kinetic study on the activation of deoxyribonuclease I by magnesium has shown that the activation curve is biphasic (the substrate being the Mg salt of bovine spleen DNA). This indicates that activation occurs at two sites on the protein. Free Mg + was required for enzyme activity, confirming that a metallo-enzyme as well as a metallo-substrate is necessary for deoxyribonuclease I activity. In contrast, it has been shown that the pyrophosphatase activity of bovine brain alkaline phosphatase depends on Mg + bound to the enzyme but not on the formation of a magnesium-substrate complex. 

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