Enzymes multiple enzyme systems

A new approach to hydroxyproline assay is a recently introduced enzymatic procedure based on the ability of a multiple-enzyme system in adapted Pseudomonas strains of bacteria to degrade hydroxyproline (R7). Also, a standard colorimetric procedure has been adapted for use in an AutoAnalyzer apparatus (G5). Automatic amino acid analyzers can be easily programmed to isolate hydroxyproline from its more usual place in the shoulder of the aspartic acid peak (see M15). 

In Sect. 7.4.3, the reduction of dihydroxyphenylpyruvic acid (DHPP) to dihydrox-yphenyllactic acid (DHPL) was used as an example for discussion of the kinetics of multiple enzyme systems (Eq. (49)). The rate equations for the reduction reaction of DHPP to DHPL (v>i) and the regeneration of PEG-NAD+ to PEG-NADH (v2) have been introduced (Eqs. (50) and (51)). 

Picloram has multiple biochemical effects in the plants. In spite of the many investigations carried out so far, the exact mode of action is unknown. The main action is undoubtedly the effect exerted on nucleic acid synthesis and metabolism and, thus, on cell protein synthesis. Moreover, picloram probably affects to a different extent several enzymes and enzyme systems. 

Homogenization of Tissues 3 Differential Centrifugation Technique 3 Separation of Cellular Components Intracellular Distribution of Biochemical Components 7 Distribution of Basic Cellular Constituents Reference Compounds Distribution of Multiple-Enzyme Systems... 

Before studying the intracellular distribution of various biochemical compounds and multiple enzyme systems, the preparative methods of cellular organelles should be described and evaluated [1-11]. 

This review of the biochemical composition of the cytoplasmic fractions is divided into three parts (1) the distribution of basic cellular constituents (nucleic acids, protein, nitrogen, and phospholipids) (2) the study of those constituents in a specific cytoplasmic organelle and (3) the intracellular distribution of multiple-enzyme systems. 

As pyruvic acid decarboxylation constitutes the link between glycolysis and the Krebs cycle, a-ketoglutaric decarboxylation divides the reactions involving 6-carbon acids (citrate, isocitrate, and oxalosuccinate) and those involving 4-carbon acids (succinate, fumarate, and malate). The analogy between the two reactions is not restricted to their role in intermediate metabolism, but extends also to the mechanism of action of the two multiple-enzyme systems. In a-ketoglutaric decarboxylation, the overall reaction leads to the formation of CO2 and succinate. CoA, NAD, thiamine, lipoic acid, and magnesium are requirements for this multiple-enzyme system activity. 

As is so often the case with multiple-enzyme systems, in beginning studies investigators needed a particulate enzyme preparation to demonstrate the reaction. Later, however, a soluble enzyme preparation became available, and it was demonstrated that the various steps of the reaction are performed by a high molecular weight compound to which thiamine pyrophosphate and lipoic acid are attached. The detailed mechanism of the reaction is far from understood it is not even known how many enzymes are involved. In bacteria, the complex has been resolved into two separate fractions named A and B, and neither fraction was found to be active by itself. In other words, a-ketoglutarate is oxidized and decarboxylated only if the A and B fractions are recombined. 

The various catalytic properties of aconitase, for example, might be due to the presence of several proteins so similar in many respects that separation is difficult. Nevertheless, as we have already pointed out, the two catalytic properties of aconitase have been separated in A. niger. Furthermore, the claim that these various catalytic properties are due to a single protein because the preparation is declared pure on the basis of ultracentrifugal and electrophoretic analysis is unjustified, because these methods demonstrate only that the protein components of the material under study are similar in size and mobility. Most protein chemists would require more severe criteria of purity, such as end-group analyses. This line of reasoning applies a fortiori to the multiple-enzymes systems. 

A second multiple-enzyme system present in liver cytosol catalyzes the conversion of malonyl CoA to fatty acids. Among the requirements for the reactions are acetyl-CoA and NADPH the products are palmitic acid, CO2, CoASH, and NADP. 

The conversion of malonyl CoA to palmitic acid is a complex reaction that involves a multiple-enzyme system in the course of which several intermediates are formed. Much of our knowledge of the intricacies of that reaction stems from work by Wakil and Lynen and their associates. The available evidence suggests that the formation of palmitic acid involves the intermediate condensation of acetyl-CoA and malonyl CoA. This reaction is accompanied by the release of CO2 and leads to the formation of a jS-keto acid, which is in turn reduced to the j8-hydroxy acid. The elimination of one molecule of water from the hydroxy acid yields the a-jS-unsaturated fatty acid, which is saturated by further reduction. 

Although there is no disagreement concerning the appearance of the endoplasmic reticulum on thin section, the tridimensional reconstruction of this planar structure is debated. The controversy is easier to understand if the proposed roles of the endoplasmic reticulum in biology are kept in mind. Among these hypothetical roles are (1) the endoplasmic reticulum provides compartments within the cytoplasm (2) it separates multiple-enzyme systems or separates the enzyme from its substrate (3) it provides a support for enzymes and substrates, thereby facilitating reaction and (4) it furnishes an intracellular circulatory system in connection with the exterior, which promotes rapid diffu-... 

The egg and the embryonic cell are well endowed with bioenergetic pathways. The multiple-enzyme systems involved in glycolysis, the hexose monophosphate shunt, the Krebs cycle, the electron transport chain, and oxidative phosphorylation have all been found in the vertebrate embryo. In the embryonic and in the mature cell, oxidation through the Krebs cycle, electron transport, and coupling of oxidation and phosphorylation occur in mitochondria. The chemical energy provided by these pathways is needed for normal development because if either glycolysis, Krebs cycle, or electron transport chain inhibitors are administered in vivo or added to explanted chick or sea urchin embryos, embryonic development is arrested. 

In addition to hemoglobin, the red cell contains 65% water (see Tables 6-2 and 6-3). Thus, except for water, hemoglobin is by far the most important biochemical. In spite of the large amounts of hemoglobin found in the red cell, one should not underestimate the importance of the remaining 3-7%, which is made up mainly of proteins and lipids. The proteins provide several multiple-enzyme systems whose functions are vital to the erythroblast. For these reasons, the histo-chemical and biochemical properties of the red cell at various stages of maturation will briefly be reviewed. 

Effect of Insulin on Individual Enzymes and Multiple-Enzyme Systemes Hexokinase Theory... 

PHA can be synthesised using proper catalysts (i.e., zinc- or aluminium-based catalyst) with water as the cocatalyst. This in vitro system has been shown to be possible using the PHA synthases purified from various sources [141, 179-181]. It is possible to produce homopolymers and copolymers containing 3HB, 3HV, 4-hydroxyvalerate and 3-hydroxydecanoate [90]. Multiple-enzyme systems have been developed that can utilise cheaper substrates as well as recycle expensive cofactors such as coenzyme A. Nevertheless, the in vitro systems are still expensive to use in order to produce PHA for applications as commodity plastics. Furthermore, hazardous organic solvents are generally required to achieve high enzymatic activity. Present studies focus on the replacement of these organic solvents with supercritical fluids [182] and ionic liquids [183]. 

Multiple enzyme systems, where one enzyme produces an electrochemically inactive product that is consumed as a substrate by another enzyme to form an active product, have been successfully used to extend enzyme selectivity. The selectivity of immunochemical systems has been employed by implementation of enzyme-linked assays. Direct coupling of redox relay centers of enzymes to conductive electrodes has been achieved by a technique known as molecular wiring and avoids the indirect analysis of products of enzyme-substrate reactions. This fast and sensitive technique measures current flow and is commercially available. 

---