Isozyme kinetics

Pyruvate kinase (PK) is one of the three postulated rate-controlling enzymes of glycolysis. The high-energy phosphate of phosphoenolpyruvate is transferred to ADP by this enzyme, which requires for its activity both monovalent and divalent cations. Enolpyruvate formed in this reaction is converted spontaneously to the keto form of pyruvate with the synthesis of one ATP molecule. PK has four isozymes in mammals M, M2, L, and R. The M2 type, which is considered to be the prototype, is the only form detected in early fetal tissues and is expressed in many adult tissues. This form is progressively replaced by the M( type in the skeletal muscle, heart, and brain by the L type in the liver and by the R type in red blood cells during development or differentiation (M26). The M, and M2 isozymes display Michaelis-Menten kinetics with respect to phosphoenolpyruvate. The Mj isozyme is not affected by fructose-1,6-diphosphate (F-1,6-DP) and the M2 is al-losterically activated by this compound. Type L and R exhibit cooperatively in... 

Enzymes that perform the same catalytic function are known as homologous enzymes and fall into two classes. Heteroenzymes are derived from different sources and although they catalyse the same reaction they show different physical and kinetic characteristics. The hydrolytic enzyme a-amylase (EC 3.2.1.1) is found in the pancreatic secretion in man and is different from the enzymes of the same name which are derived from bacteria or malt. Iso-enzymes, sometimes referred to as isozymes, are different molecular forms of the same enzyme and are found in the same animal or organism although they often show a pattern of distribution between tissues. 

Some of the results obtained by differential centrifugation showed enzyme distribution between different cell fractions which were difficult to interpret. Enzymes like carbamoyl phosphate synthase or isocitrate dehydrogenase were found both in mitochondria and in the soluble fraction of the cell. This led to detailed kinetic studies with purified enzymes which indicated there might be populations of enzymes with slightly different properties (isozymes) catalyzing similar reactions in different compartments or in different cell types. Variations in kinetic behavior appeared to tailor the enzyme appropriately to the particular compartment or cell where the reaction took place. 

Lignin peroxidase, secreted by the white-rot fungus Phanerochaete chrysosporium in response to nutrient deprivation, catalyzes the H202-dependent oxidation of non-phenolic aromatic substrates. The present report summarizes the kinetic and structural characteristics of lignin peroxidase isozymes. Our results indicate that the active site of lignin peroxidase is more electron deficient than other peroxidases. As a result, the redox potential of the heme active site is higher, the heme active site is more reactive and the oxycomplex is more stable than that of other peroxidases. Also discussed is the heme-linked ionization of lignin peroxidase. 

Isozymes are also a common presence in enzyme preparations and they can often be detected via polyacrylamide gel electrophoresis. The detected presence of isozymes may result in the need for further purification steps and the kinetic characterization of each isozyme. It may be necessary to use nondenaturing electrophoretic procedures to separate the different isozymes. See Isozymes Enzyme Concentration... 

Haldane relationships can also be useful in characterizing isozymes or the same enzyme isolated from a different source. Reactions catalyzed by isozymes must have identical equilibrium constants, but the magnitudes of their kinetic parameters are usually different (e.g., the case of yeast and mammalian brain hexokinase ). Note that the Haldane relationship for the ordered Bi Bi mechanism is = Hmax,f p i iq/(f max.r ia b)- This same... 

In order to prove that specific isozymes are, in fact, involved in lignin formation, each must first be purified to homogeneity and subjected to appropriate immunochemical and kinetic investigations. Some cautious optimism for the resolution of this problem can be hoped for, since an antibody of the isozyme from Nicotiana tabacum has been obtained and its cDNA cloned (75). It is to be hoped that the following can now be determined ... 

The unit is mM. Each value is connected to a substrate name. The commentary gives, if available, information on specific reaction condition, isozymes or presence of activators. The references for values which cannot be expressed in mM (e.g. for macromolecular, not precisely defined substrates) are given in Additional Information. In this field we also cite literature with detailed kinetic analyses. 

The four forms of hexokinase found in mammalian tissues are but one example of a common biological situation the same reaction catalyzed by two or more different molecular forms of an enzyme. These multiple forms, called isozymes or isoenzymes, may occur in the same species, in the same tissue, or even in the same cell. The different forms of the enzyme generally differ in kinetic or regulatory properties, in the cofactor they use (NADH or NADPH for dehydrogenase isozymes, for example), or in their subcellular distribution (soluble or membrane-bound). Isozymes may have similar, but not identical, amino acid sequences, and in many cases they clearly share a common evolutionary origin. 

Isotope incorporation 489 Isotope exchange kinetics 47, 235, 236,240,563-569 Isozyme 461 -465... 

The enzyme, which has been isolated from many species, is a tetramer of Mr 140 000. The two forms of the enzyme, the H4, predominating in heart muscle, and the M4, predominating in skeletal muscle, give rise to a family of isozymes.46,47 The amino acid composition of the polypeptide chain that constitutes the M4 form is significantly different from that of the H4 form, and the two have different kinetic properties. Despite this, the sites for the association of the subunits had to... 

Inhibition of the chicken breast enzyme by rabbit antisera for chicken breast enzyme the lack of effect on the chicken brain, heart, or erythrocyte enzyme and the differences in substrate specificity exhibited by the brain and breast muscle enzyme are consistent with at least two isozymes of chicken 5 -AMP aminohydrolase (123). Isozymic patterns, while perhaps implied by differences in certain kinetic pa-... 

Scott (80) purified red cell acid phosphatase of homozygous types A and B by using ammonium sulfate and DEAE-cellulose chromatography. The relative activity of these isozyme preparations was the same when tested with a number of substrates. Type B enzyme showed small kinetic... 

There are several known effects of BHA treatment which have been proposed to reduce the levels of reactive metabolites of BP which bind to DNA. It has been demonstrated that BHA feeding alters the microsomal mixed-function oxidase system in mice and rats (7,17-19). Several studies suggest that BHA treatment does not decrease the amounts of BP-7, 8-diol formed (7,20,21) whereas there is some indirect evidence that BHA treatment alters the metabolism of BP-7,8-diol (3,20) An induction by BHA of an lsozyme(s) of cytochrome P-450, which has kinetics of metabolism of BP-7,8-diol different than that of the constitutive isozyme(s), could account for the BHA-induced shifts in the dose-response curve for BPDEI-DNA adduct levels (15). 

ISOZYME One of several forms of an enzyme that have similar catalytic activities, but differ in kinetic properties and often in electrophoretic mobility. 

Glumoff T, Harvey PJ, Molinari S et al (1990) Lignin peroxidase from Phanerochaete chrysosporium. Molecular and kinetic characterization of isozymes. Eur J Biochem 187 515-520... 

SA in Table II - nmoles/hr/mg protein and N.D - not detectable. Tobacco, soy cultures and barley seedlings were the best source of ALS, both in terms of specific activity and total units. The enzyme preparations from all sources were unstable in buffer solutions in spite of protective thiol agents. The inactivation of ALS in the crude extract of tobacco showed a distinct biphasic kinetics, implying the presence of at least two isozymes (unpublished observations). The presence of two ALS genes in tobacco (29) and at least three in microorganisms (18) has also been noted by other workers. ALS from barley was most amenable to purification. Table III gives a profile for the rapid purification of this enzyme with high recovery. 

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