Enzymic and Physico-chemical Studies

Following the discovery of methods for the preparation of nucleic acids, Iwanoff studied the action of various molds on desoxyribose-nucleic acid, and the results led to his postulating the existence of a specific nucleoclastic enzyme, or enzymes, which he termed nuclease.  

Levene and Dillon discovered that the gastro-intestinal secretions of the dog contain a polynucleotidase and a non-specific nucleotidase. Whereas uridylic acid is the most stable of the natural ribosenucleotides as regards hydrolysis with mineral acids, it is more readily dephosphory-lated by nucleotidase than is any other nucleotide. 

In 1912, Jones - isolated, from pig s pancreas, a supposed nucleinase which was stable to heat. On treating a solution of ribosenucleic acid with this enzyme, the acid underwent a change which was presumed to be fission to the individual mononucleotides but no change in the acidity of the solution was detected. Jones interpreted this as indicating that 

Levene mentioned in 1931 that he had been unable to hydrolyze the polynucleotide to the mononucleotides with this enzyme, and in 1937 Dubos continued the study of the enzyme. Schmidt and Levene came to the conclusion that the phenomenon is one of depolymerization of polytetranucleotides to the tetranucleotide state. They were unable to detect any reaction-product of small enough molecular size to diffuse through cellophane membranes, and found the freezing point of the solution remained unchanged during action of the enzyme. However, a mixture of the four mononucleotides dialyzed rapidly through cellophane and caused a considerable depression of the freezing point. 

There would appear to be at least two possible explanations for the discrepancy between the results of Schmidt and Levene and those of Kunitz regarding the dialyzability of the end-products. The first is that Schmidt used as enzyme a boiled solution of commercial pancreatin, whereas Kunitz employed crystalline ribonuclease which had not been exposed to heat. The second possibility has been pointed out by Loring and Carpenter. They consider that dialysis in the presence of the enzyme preparation, which probably contained an appreciable amount of inert protein, is not comparable with dialysis of a mixture of the four mononucleotides in the absence of the enzyme preparation. Simple control experiments would surely serve to establish the truth or falsity of these speculations. 

Allen and Eiler discovered that whereas the polymerized acid had four primary phosphoric dissociations per tetranucleotide unit, after action of the crystalline enzyme one secondary phosphoric dissociation per tetranucleotide unit made its appearance (in addition to the four primary dissociations). They decided that this behavior is compatible with Levene s open-chain formula (VII) for the tetranucleotide provided that, in the polymer, the secondary hydroxyl of one phosphoryl group per tetranucleotide unit is involved in the polymerization. They also pointed out that it could be interpreted as the opening of the cyclic structure proposed by Takahashi (VIII). 

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Based on X-ray crystallographic results and other studies, the most important catalytic residues appear to be Lys-258 and Tyr-70. This is because the a-proton of the substrate amino acid must be removed by the action of the catalytic base at the beginning of the catalytic process, and this step takes place stereospecifically at the 57-plane of the PLP ring. Lys-258 and Tyr-70 are the only residues which are located within van der Waals contact distance from the a-proton and are able to participate in the proton abstraction. The results of various chemical modification and physico-chemical studies on wild-type enzyme, and high resolution X-ray crystallographic studies could not clearly define Lys-258 and Tyr-70 as a catalytic base. Therefore, mutagenesis studies on Lys-258 and Tyr-70 were used to define the roles of these residues. 

A fundamental problem associated with studies of enzyme mechanism is that even the purest samples of these catalysts contain relatively large amounts of impurities such as inactive protein and water. Moreover, samples are often solutions or suspensions of the enzyme in aqueous media and the basic analytical technique of accurate weighing is thus not appropriate to produce a standard solution of enzyme which meets the criteria normally demanded in physico-chemical studies of mechanism. 

It is now well established that the catalytic properties of a wide variety of enzymes remain intact in organic solvents (11-13). These findings imply that proteins may also retain their native struetures when lyophilized and dispersed in organic solvents. Evidence has been obtained that crystallized proteins have essentially the same structure in water and organic solvent (14,15). In the lyophilized state, proteins are also in a nonaqueous environment and it is expected their physico-chemical properties will differ from that in solution, as the dynamic conformational equilibria that exits in solution will be absent. Some physico-chemical studies indicate that the structure of the lyophilized state is very similar to that in solution (16-18), while others indicate that there is some limited but reversible conformational change (19-24). There are likely to be... 

The impact of different factors affecting the process of enzyme entrapment and its reflection on the activity of the immobilized enzyme, its physico-chemical characters and its kinetic parameters have been studied and the obtained results are given. 

Primary differentiation of the cytoplasm of the oocyte of this type has now been studied not only in relation to the external characteristics of the membrane or incorporation of the cytoplasm, but also in relation to many biochemical and physico-chemical indices, such as redox potential, content of lipids, enzymes, and RNA, rate of protein synthesis, and so on, in many species of animals. It is impossible even to make a short examination of the many different variants of ooplasmic segregation. However, it is extremely important, because it determines the primary directions of differentiation. 

Despite the extensive use to which photochemical oxidations of proteins have been put very little attention has been given to identifying the end products of the photoreactions. Apart from methionine which is converted to methionine sulfoxide and more slowly to its sulfone, the fate of the other amino acid residues of irradiated proteins remains largely unknown. On the basis of limited chemical studies, it is clear, however, that photoreactions usually lead to a mixture of products. The multiplicity of products formed by photooxidation of enzymes will undoubtedly limit the utility of the technique. There are in fact numerous examples from work dealing with the chemical modification of enzymes which indicate that modification of a particular residue with different chemical reagents leads to enzyme derivatives with different biological and physico-chemical properties. 

The polygalacturonase was studied and this enzyme was secreted constitutively both under anaerobic or aerobic conditions. A few physico-chemical properties of the secreted PG have been determined (PM, PI) [2],... 

Synthetic substrates allow rapid determination of the catalytic constants of an enzyme. Nevertheless, it is known that the environment of the peptide bond depends largely on physico-chemical conditions of the applied media, and imposed steric hindrance. Since these parameters are important, the hydrolysis of purified (3-casein was studied at different pHs. The kinetic analysis revealed that the mutant conserved the native trypsin capacity to hydrolyze peptide bonds containing arginyl and lysyl residues. The optimal pH of activity changed considerably according to the mutation. 

The comparison of exopolyphosphatases from different cell compartments of the yeast S. cerevisiae suggests that they are a typical example of compartment-specific enzymes. The latter differ from each other in their physico-chemical properties, substrate specificity, response to changing cultivation conditions, and presumably, in the functions and ways of regulation. The compartment-specificity of exopolyphosphatases should be taken into account in the study of PolyP metabolism in the eukaryotic cell. 

During the 1950s and 1960s, studies focussed on the milk lipase system, the mechanism of its activation and the physico-chemical properties of the enzyme(s) involved. From these studies it was concluded that more than one lipase was present and several attempts to purify a milk lipase were reported. Jensen (1964) reviewed much of this work. 

Mass spectroscopy, in combination with other physico-chemical and biochemical methods, promises to be an effective tool for fruitful study of the structure and action mechanism of enzymes. 

Immobilization of the enzymes to sohd surface induces stractirral changes which may affect the entire molecule. The study of conformational behavior of enzymes on solid surface is necessary for better understanding of the irtrmobilization mechanism. However, the immobilization of enzymes on alginate beads is generally rapid, and depends on hydrophobic and electrostatic interactions as well as on external conditions such as pH, temperature, ionic strength, and nature of buffer [11,12], Enzymes dena-turation may occur under the irtfluence of hydrophobic interactions, physico-chemical properties of the alginate beads or due to the intrinsic properties of the enzyme. 

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