Enzyme multiple form

Although the first study of exopolygalacturonases from carrots [11] indicated the presence of multiple forms of this enzyme based on the three present pH optima, the latter studies supported the idea of one form of exopolygalacturonase [2-4]. The present study deals with the whole spectrum of multiple forms of exopolygalacturonase, with forms described sooner and with forms found only now. 

Candida boidinii is a further yeast producer of pectic enzymes complex. The production is induced by the presence of pectin as a C-source in the medium the primary methabolic path is the utilization of methanol and the secondary the utilization of pectate chains. The pectic enzymes were bound on the cell walls or released on the cultivation medium. The main enzyme of pectic complex, polygalacturonase, was briefly characterized and the possibility to influence the production of its multiple forms discussed. 

The activity of the main enzyme of pectic enzymes complex, polygalacturonase, was dependent on the pH of the cultivation media the highest activity was reached at pH 3.51 (natural pH of pectin medium), the activity decreased to 70% and 20% by the cultivation at pH 5.49 and pH 7.01, respectively. The isoelectric focusing patterns showed the production of polygalacturonase multiple forms with isoelectric points varying from 3.5 to 7.5 (Fig. 3 A,B) with the possibility to influence their production with the change of the C-source and pH of the cultivation media. 

Triose phosphate isomerase (TPI) catalyzes the interconversion of glyceralde-hyde-3-phosphate and dihydoxyacetone phosphate and has an important role in glycolysis, gluconeogenesis, fatty acid synthesis, and the hexose monophosphate pathway. Red blood cell TPI activity measured in vitro is approximately 1000 times that of Hx, the least active glycolytic enzyme. TPI is a dimer of identical subunits, each of molecular weight 27,000, and does not utilize cofactors or metal ions. Posttranslational modification of one or both subunits may occur by deamidination, resulting in multiple forms of the enzymes and creating a complex multibanded pattern on electrophoresis. 

Fungal laccases (benzenediokoxygen oxidoreductase, EC 1.10.3.2) belong to the multicopper blue phenoloxidases. They comprise glycosylated proteins expressed in multiple forms and variable molecular weight, ranging from 59 to 110 kDa. Laccase is expressed as multiple constitutive and induced isoenzymes [30, 64]. The enzyme contains four copper atoms (Cu), in different states of oxidation (I, II, III) [65], which play an important role in the catalytic mechanism. Laccase oxidizes different compounds while reducing O2 to H20, a total reduction of four electrons. 

Multiple forms of heterotrimeric G proteins exist in the nervous system. Three types of heterotrimeric G protein were identified in early studies. G termed transducin, was identified as the G protein that couples rhodopsin to regulation of photoreceptor cell function (see Ch. 49), and Gs and G were identified as the G proteins that couple plasma membrane receptors to the stimulation and inhibition, respectively, of adenylyl cyclase, the enzyme that catalyzes the synthesis of cAMP (see Ch. 21). 

Protein kinase C (PKC) comprises the other major class of Ca2+-dependent protein kinases and is activated by Ca2+ in conjunction with DAG and phosphatidylserine (discussed in Ch. 20). Multiple forms of PKC have been cloned, and the brain is known to contain at least seven species of the enzyme. The variant forms of PKC exhibit different cellular distributions in the brain and different regulatory properties. For example, they differ in the relative ability of Ca2+ and DAG to activate them some require both Ca2+ and DAG, whereas others can be activated by DAG alone, apparently without an increase in cellular Ca2+ concentrations. However, these enzymes show similar substrate specificities and, as a result, are often considered isoforms. 

Many iso-enzymes are hybrids of a limited number of sub-units. Some enzymes show multiple forms owing to increasing levels of polymerization of a single sub-unit these should not really be called iso-enzymes, because they do not have any genetic difference between the different forms. Cholinesterase, for instance, shows five forms consisting of a single sub-unit existing as monomers, dimers, trimers, tetramers and pentamers. 

The marine environment acts as a sink for a large proportion of polyaromatic hydrocarbons (PAH) and these compounds have become a major area of interest in aquatic toxicology. Mixed function oxidases (MFO) are a class of microsomal enzymes involved in oxidative transformation, the primary biochemical process in hydrocarbon detoxification as well as mutagen-carcinogen activation (1,2). The reactions carried out by these enzymes are mediated by multiple forms of cytochrome P-450 which controls the substrate specificity of the system (3). One class of MFO, the aromatic hydrocarbon hydroxylases (AHH), has received considerable attention in relation to their role in hydrocarbon hydroxylation. AHH are found in various species of fish (4) and although limited data is available it appears that these enzymes may be present in a variety of aquatic animals (5,6,7,8). 

Fig. 4.1. Fundamentals of the ubiquitin system. Adapted from Ref [5]. Figure 4.1 shows the fundamentals of the ubiquitin system. (1) Ubiquitin is synthesized in linear chains or as the N-terminal fusion with small ribosomal subunits that are cleaved by de-ubiquitylating enzymes to form the active protein. Ubiquitin is then activated in an ATP-dependent manner by El where a thiolester linkage is formed. It is then transthiolated to the active-site cysteine of an E2. E2s interact with E3s and with substrates and mediate either the indirect (in the case of HECT E3s) or direct transfer of ubiquitin to substrate. A number of factors can affect this process. We know that interactions with Hsp70 can facilitate ubiquitylation in specific instances and competition for lysines on substrates with the processes of acetylation and sumoylation may be inhibitory in certain instances. (2) For efficient proteasomal targeting to occur chains of ubiquitin linked internally through K48 must be formed. This appears to involve multiple...

As described in the previous section, this strain produced significant amounts of three extracellular P-mannanases and a cell-associated P-mannosidase. The three P-mannanases differed in several enzymatic properties including optimum pH for enzyme action, optimum temperature, pH stability, thermal stability, isoelectric point and molecular weight. To elucidate the genetic basis for production of multiple forms,... 

Enzyme Nomenclature. Recommendations (1992) Academic Press, New York Nomenclature of multiple forms of enzymes J. Biol Chem. (1977) 252, 5939-5941 Catalytic activity Units of enzyme activity Eur. J. Biochem. (1979) 97, 319-320 Symbolism and terminology in enzyme kinetics Eur. J. Biochem. (1982) 128, 281-291... 

RNA polymerase II represents a multi-enzyme complex of at least 12 proteins, but whose exact composition is difficult to determine. Hiis is due to the instabUity of the holo-complex, which makes the pmification and characterization of the enzyme difficult. Furthermore, it is likely that multiple forms of RNA polymerase II exist, each of slightly different composition and performing different functions. 

The hgases involved in this type of ubiquitin ligation are organized in multiprotein complexes caUed SCF complexes (Skpl, cuUin, F box protein). In S. cerevisiae, the complex is composed of the proteins Cdc24 (an E2 enzyme), Cdc53 and Skpl, which form the core of the SCF. This core associates in a variable maimer with a further type of protein, which function as specific substrate recognition factors. A common feature of these proteins is a sequence element known as the F box (review Peters, 1998). Consequently, multiple forms of SCF complexes exist, due to this variable association. 

The frequent occurrence of sialylated enzymes, or even of multiple forms, which are sometimes tissue-dependent, with a varying number of sialyl residues as, for example, in y-glutamyltranspeptida.se (EC 2.3.2.2),456,457 is not yet fully understood. Although the activity of most of these enzymes is not influenced by removal of sialic acid,454 the activity of monoamine oxidase A (EC 1.4.3.4) of outer mitochondrial membranes of rat liver has been shown to be destroyed by treatment with sialidase438 the substrate specificity of acetylcholinesterase (EC 3.1.1.7) is altered,459 the kinetic properties of human acid and alkaline phosphatases (EC 3.1.3.1 and 3.1.3.2) are changed, and the stability of a-D-galactosidase (EC 3.2.1.22) is drastically lowered.415 In these cases, an influence of sialyl residues on the conformation of the enzyme is assumed, but awaits firm evidence. 

A new representative of a multicopper cluster in a protein is Cuz in nitrous oxide reductase. As was discussed above this enzyme contains a binuclear CuA centre as in COX. While the latter in addition has CuB in the form of a copper-heme group, N20 reductase has Cuz which is the site of dinitrogen formation from the substrate N20. Recently a central inorganic sulfide has been found as a ligand to copper and multiple forms of Cuz were detected in the enzyme from Paracoccus pantotrophus.134 More recently a tetranuclear copper cluster with X-S bridges was proposed as structure for Cuz..135... 

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. 

Nomenclature of Multiple Forms of Enzymes hitp//ww vv.cliem.qmul.ac.uk/iubmb /misc/isoen.html... 

The final form of metabolic regulation is effected by the use of iso-enzymes, which are multiple forms of an enzyme. For example, lactate dehydrogenase exists in five forms in a rat. They differ in primary structure and have different isoelectric points, but they all catalyse the reversible reduction of pyruvate to lactate. 

Isozymes. Multiple forms of an enzyme that differ from one another in one or more properties. 

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