Investigating the enzyme-substrate interactions of cytochrome c oxidase: a computer-assisted approach [abstract]

Although the enzyme cytochrome c oxidase (COX) is critical to respiration and has been studied extensively, the interactions between this enzyme and its substrate cytochrome c are still not very well understood. We employed a computer assisted approach to study these interactions. We used the Swiss-pdb v 2.5 computer programme, which measures the percentage accessibility of residues and the online server ANOLEA, which calculates the non-local energy of residues in a polypeptide chain, to analyze the respective molecular structures of cytochrome c and COX. The accessibility studies showed that, compared to the oxidized, the reduced form of cytochrome c normally had the greater proportion of more highly accessible residues: for reduced cytochrome c, 7 of the 8 residues exhibiting 55 percent accessibility were lysines. Interestingly enough, lysine 13, shown by other studies to be important for substrate binding, was not significantly accessible. For COX, neither asparate 158 nor glutamate 198 residues, reported to be important for substrate binding and catalysis, were significantly accessible either. The energy studies showed that whereas oxidized cytochrome c was a stable structure of low energy, approximatley 81 percent of the protein was reduced. For COX, a few small regions, including 4 residues in the vicinity of CuA, which functions as the enzyme's electron entry port, were of high energy. Since lysine 13, aspartate 158 and glutamate 198 are known to play important roles in enzyme-substrate interactions, it must be that these residues become more accessible when the two proteins interact. The results of the accessibility studies therefore appear to suggest that COX employs a mix of induced-fit and strain mechanisms when it binds substrate. On the basis of the energy studies, we conclude that the structure of reduced cytochrome c (the substrate) resembles a high energy transition-state intermediate and that when this protein binds and reduces oxidized COX, the structures of both proteins are stabilized. (AU)

Cytochrome c oxidase: structural evidence for conformational switching between two substrate binding sites [abstract]

Cytochrome c oxidase, the final member of the electron transport chain is critical to aerobic respiration and the absence, deficiency or malnutrition of this enzyme causes a number of myopathies and other diseases, some of which are fatal. In spite of its importance, the enzyme is still not well understood and whether one or two binding sites for its substrate cytochrome c remains unresolved. In an attempt to answer this question, we prepared 1:1 covalent enzyme-substrate complexes under conditions of low ionic strength. In addition to the `traditional' complex formed between oxidized cytochrome c and the oxidized enzyme, we prepared a new complex under `steady-state' reducing conditions. For both complexes, we investigated the ability of the covalently bound enzyme to catalyze the oxidation of unbound (exogenous) ferrocytochrome c. Whereas for the `traditional' oxidized complex, cytochrome c became bound exclusively to subunit II of the enzyme, for the `steady-state'complex, cytochrome c became bound to subunits II, VIa and VIb. Steady-state spectral analysis (400-630nm), combined with Stopped-Flow studies, confirmed that the bound cytochrome c mediated the efficient transfer of electrons from the reducing agent ascorbate to the enzyme. Additionally, pre-steady state analysis at 550nm showed haem a within both covalent complexes catalyzed the oxidation of exogenous ferrocytochroms c. The second order rate constant for these reactions was approximately 25-50 percent of those observed for controls. Our results suggests (i) that electrons are able to enter cytochrome c oxidase via two independent pathways and (ii) that during enzyme turnover the enzyme cycles between two conformers, one of with a substrate binding site at subunit II and the other wth a site at subunits VIa and VIb. Structural analysis suggests that Glu 43, Asp 64 and Glu 83 of subunit VIa and Asp 73, Asp 74 and Glu 78 of subunit VIb are residues that might possibly be involved at the latter site. (AU)

Profile of plasma and erythrocyte enzymes: whole blood 2, 3 bisphosphoglyceric acid and reduced glutathione levels in Jamaican diabetics - abstract

The metabolism of normal red cell is well adapted to protect itself from oxidative stress. The maintenance of red cell membrane, haemoglobin and enzymes containing sulfhydryl groups (-SH groups) are dependent upon the levels of reduced glutathione (GSH). Alteration in the activities of enzymes such as glutathione reductase and glutathione peroxidase will affect the levels of GSH and consequently the red cell metabolism. Acetylcholinesterase, an SH-dependent enzyme, also increases in the red cells in diabetics and is related to membrane fluidity. The level of 2, 3- Bisphosphoglycerate (2,3-BPG) which serves an important mechanism by which the body regulates its oxygen supply from haemoglobin to meet tissue demands, also increases in chronic anaemias observed in diabetics. In the present studies the activities of plasma and erthrocyte cholinesterase, and erthrocyte glucose-6- phosphate dehydrogenase were measured in 40 diabetic patients (both IDDM and NIDDM groups). The whole blood 2, 3-BPG and GSH were also estimated in these patients. All five parameters showed significant increases in diabetic as compared to non-diabetic controls. The results could point to a potential role of G6PD 2, 3-BPG GSH and choline sterase as indicators of diabetes control (AU)

Cytochrome c oxidase: a new conformational model

Cytochrome c oxidase and zinc cytochrome c were cross-linked to form a 1:1 enzyme-substrate convalent complex. The reaction was catalyzed by 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) and cytochrome c, the substrate, became attached to the binding site located at submit II of the enzyme. This covalent complex could still be reduced by unbound ferrocytochrome c, suggesting the existence of more than one substrate binding site [1]. Previously [2] we reported that the reduction of heme a triggered a major conformational change within cytochrome c oxidase. Here we propose a model which incorporates these two findings with other established features of cytochrome c oxidase. The most prominent features of this model are (i) Electrons may enter the enzyme via both heme and CUa. Thus cytochrome oxidas may be regarded as having two substrate binding sites, one near heme a and the other near CUa. (ii) At any instant however, only one site is normally available for the binding of substrate. (iii) Subsequent to binding, a substrate molecule transfers an electron to the redox center at the site; reduction of the redox centre triggers a conformational transition which abolishes that binding site and simultaneously leads to the formation of an adjacent substrate binding site near the other redox centre. (iv) The two sites possess approximately equivalent affinity for substrate(AU)

Bio-specific purification of folate-requiring enzymes - abstract

Despite the use of a variety of anti-folate agent in chemotherapy, detailed understanding of the metabolism of folates is scant due largely to the instability of folate coenzymes in vitro, and the low levels of the enzymes involved in vivo. Commercially available tetrahydrofolate preparations contain at least 25 percent unspecified impurities, and are racemic mixture of biologically active and inactive isomers. The object of this study was to investigate the application of purified substrates in the isolation of folate-metabolising enzymes by affinity chromatography. Methylenetetrahydrofolate, which is both oxygen - and photo-labile, was prepared by reducing folic acid with borohydride and subsequent reaction with formaldehyde. The biologically active isomer, d-methylenetetrahydrofolate, was isolate by ion-exchange chromatgraphy, with a yield of 35 percent from folic acid. Methylenetetrahdrofolate reductase, an allosteric enzyme regulating the biosynthesis of S-adenosylmethionine, was partially purified from rat liver by ion-exchange chromatography and applied to a column of the affinity matrix procion red HE-3B - sepharose. The loaded column was washed with buffer to remove unbound proteins and active enzyme was bio-specifically eluted with a gradient of d-methylenetetrahydrofolate with greater than 90 percent recovery of activity. Hereditary deficiency of this enzyme has been implicated in cases of schizophrenia and homocystinuria. The quantitative determination of enzyme levels in vivo, and kinetic data obtained from purified preparations in vitro are prerequisites for the understanding of metabolic fluxes in the intact animal. The technique investigated in this study may facilitate analysis of other folate-requiring enzymes (AU)

Serum enzyme changes in tetanus

The activities of seven serum enzymes were followed in ten cases of tetanus. Enzymes present in the sketal muscle might be useful in indicating the severity of the disease and in following the course of therapy. It might also be useful to determine the CPK-MB isoenzyme to detect any mycordial involvement. Abnormal liver function tests were also observed. These resolves when the patients recovered. Cholinesterase activity has not been found to be useful either in determining the severity of the disease or in following the course of therapy (AU)

A controlled trial of hycanthone and placebo in schistosomiasis mansoni in St. Lucia

In a blind trial, 32 schistosomiasis mansoni patients (mean age, 12.03 years) in St. Lucia were given a single intramuscular dose (2.5 mg/kg of body weight) of either hycanthone or a vitamin placebo. The principal side effect, vomiting, was limited to four of the 16 patients given hycanthone; three patients in the hycanthone group and two of the 16 given placebo complained of abdominal pain. Daily enzyme determinations suggest that the mild elevations of serum glutamic oxaloacetic transaminase, lactic dehydrogenase and creatine phosphokinase observed in both groups were due to muscle injury at the injection site. Six months after treatment, only the hycanthone group had a marked reduction in egg excretion (99 percent), with 7/16 then excreting no eggs. The hycanthone group had a greater but statistically insignificant decrease in symptoms at six months. Of 30 patients with hepatomegaly, only the 16 in the hycanthone group showed a statistically significant decrease in liver size at six months; of seven patients with splenomegaly, the two in the hycanthone group and 1/5 in the placebo group showed a decrease in spleen size. All patients gained in weight during the study, with the mean increase for the hycanthone group being greater by 1.2 kg. At the conclusion of the study, the placebo group patients were treated with hycanthone (AU)

In-born errors of metabolism

The causes of the majority of in-born errors of metabolism have yet to be fully elucidated, further knowledge of the mechanisms of these lethal metabolic disorders is necessary in order to employ adequate therapy. A very interesting line of therapy was observed in a recent paper by Schneck et al who diagnosed Tay Sach's disease after ammniocentesis, and subsequently terminated the pregnancy. Prevention to my mind could in the future be the primary aim of therapy in certain of these lethal disorders rather than one subjecting himself to the pros and cons and the psychological trauma of euthanasia. It would also be interesting to see how far prenatal diagnosis could go to aid the prevention of lethal haemoglobinopathies. The concept of prevention within this concept without doubt poses a serious ethical problem. (Summary)

The measurement of total protein synthesis and catabolism and nitrogen turnover in infants in different nutritional states and receiving different amounts of dietary protein

Total nitrogen turnover and the rates of synthesis and catabolism of total body protein were measured in infants, by means of a constant intragastric infusion of [15N] glycine. Experimental evidence is presented to support the assumption that amino acids from food and from tissue protein catabolism are indiscriminately handled by the body, and that [15N] glycine is a valid tracer for the mixture of total amino-N. N turnover, synthesis and catabolism of protein were all significantly higher in the malnourished as compared to the recovered infant. Net protein synthesis was the same in the two states, and a greater proportion of the N entering the pool was synthesized into protein in the malnourished infant. The rate of total protein synthesis in recovered infants was about 6g per kg body weight per day and was the same in infants receiving a low protein or a high protein intake. However, catabolism was significantly increased and there was greater utilization of N for protein synthesis in infants on a low protein diet. The mechanisms of adaptation to infantile malnutrition and to a low protein diet may be brought about through changes in amount or activity of enzymes concerned with amino acid metabolism. (AU)

Senescence in the placenta: an enzyme histochemical study

There is a variety of experimental and clinical evidence available suggesting that the placenta undergoes age changes during the last 4-6 weeks of pregnancy, which are associated with corresponding reduction in its functional capacity. Although the anatomical criteria for ageing in the placenta are not by any means universally accepted, a general pattern is beginning to emerge. During the last trimester, the sycytiotrophoblast appears to undergo progressive degeneration so that the cytoplasm becomes gradually thinner, and the nuclei are displaced into small clusters referred to as "syncytial knots". Parallel with this change, there is occasionally a proliferation of the cytotrophoblast which has usually undergone gradual reduction in amount during the middle trimester, but appears to regenerate under the stimulus of anoxia. The connective tissue stroma of the villi becomes progressively denser and more collagenous, occasionally undergoing hyaline change. The vessels of the stroma meanwhile, tend to undergo narrowing of their lumen. The microscopical changes which have been described, are of a fairly gross nature, and they may represent the end results of a progressive degenerative process. In order to determine the earliest changes occurring in the villi of the placenta, which it was assumed would probably be of a metabolic nature, an enzyme histochemical study was undertaken. Multiple biopsies were obtained from 92 placentas representing various normal and abnormal pregnancies at different stages of gestation. The biopsies obtained from each placenta were made into composite blocks and snap-frozen using a dry ice-acetone mixture. These composite blocks were then stored at minus 20 degrees C. until processed. Eight micron sections of each composite block were obtained by cutting in a Pears ctyostat. The results can be briefly summarised as follows: (1) Alkaline Phosphatase and glucose-6-phosphatase showed a rising concentration in the trophoblast with advancing pregnancy. (2) Acid Phosphatase within the trophoblast showed an apparent decrease in amount in late pregnancy. (3) Lactate Dehydrogenase; Malate Dehydrogenase; Alpha-glycero-phosphate Dehydrogenase; and glucose-6-Phosphate Dehydrogenase were present in the trophoblast throughout pregnancy without showing any apparent change in concentration. (4) Succinate Dehydrogenase could not be found in the trophoblast at any time, occurring only in the decidua underlying the placental bed. Leucine Amino-peptidase showed a patchy distribution throughout the connective tissue stroma of the chorionic villi and within the walls of the larger blood vessels. On the basis of these findings, it is suggested that a rising content of alkaline phosphatase and glucose-6-phosphatase, associated with a falling concentration of acid phosphatase, constitute histochemical criteria on which a diagnosis of placental senescence can be based(AU)

Placental insufficiency investigated by enzyme histochemistry

The enzyme histochemistry of 92 placentae representing various normal and abnormal pregnancies at different stages of gestation was studied. Alkaline phosphatase and glucose-6-phosphatase showed a rising concentration in the trophoblast with advancing pregnancy. Acid phospatase within the trophoblast showed an apparent tendency to decrease in amount in late pregnancy. Lactate dehydrogenase, malate dehydrogenase, alpha-glycero-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase were present in the trophoblast throughout pregnancy without any apparent change in concentration. Succinate dehydrogenase could not be found in the trophoblast at any time, occurring only in the decidua underlying the placental bed. Leucine amino-peptidase showed a patchy distribution throughout the connective tissue stroma of the chorionic villi and within the walls of the larger blood vessels. It was suggested that a high concentration of alkaline phosphatase and glucose-6 phosphatase associated with a low concentration of acid phosphatase constituted the histochemical parameters of placental insufficiency (AU)

Further observations of enzymes in the human liver in malnutrition

In malnourished liver, although simple enzyme systems are on the whole well maintained, more complex functions may be damaged. These functions are of obvious importance for the vitality of the cell. It is necessary first to confirm these findings, and then to study the mechanism. Decreased phosphatide formation might impair the integrity of the mitochondrial membrane, but so far the type of test that we have used has not confirmed this(AU)