Enzyme catalysis in microgravity: steady-state kinetic analysis of the isocitrate lyase reaction

Two decades of research in microgravity have shown that certain biochemical processes can be altered by weightlessness. Approximately 10 years ago, our team, supported by the European Space Agency (ESA) and the Agenzia Spaziale Italiana, started the Effect of Microgravity on Enzyme Catalysis project to test the possibility that the microgravity effect observed at cellular level could be mediated by enzyme reactions. An experiment to study the cleavage reaction catalyzed by isocitrate lyase was flown on the sounding rocket MASER 7, and we found that the kinetic parameters were not altered by microgravity. During the 28th ESA parabolic flight campaign, we had the opportunity to replicate the MASER 7 experiment and to perform a complete steady-state analysis of the isocitrate lyase reaction. This study showed that both in microgravity and in standard g controls the enzyme reaction obeyed the same kinetic mechanism and none of the kinetic parameters, nor the equilibrium constant of the overall reaction were altered. Our results contrast with those of a similar experiment, which was performed during the same parabolic flight campaign, and showed that microgravity increased the affinity of lipoxygenase-1 for linoleic acid. The hypotheses suggested to explain this change effect of the latter were here tested by computer simulation, and appeared to be inconsistent with the experimental outcome.     

The catalytically active domain in the A subunit of calcineurin

Calcineurin (CaN) is a heterodimer composed of a catalytic subunit A (CaNA) and a regulatory subunit B (CaNB). We report here an active truncated mutation of the rat CaNAdelta that contains only the catalytic domain (residues 1-347, also known as a/CaNA). The p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were higher than that of CaNA. Both p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were unaffected by CaM and the B-subunit; the B-subunit and CaM have relatively little effect on p-nitrophenyl phosphatase activity and a crucial effect on protein phosphatase activity of CaNA. Mn2+ and Ni2+ ions effeciently activated CaNA. The Km of a/CaNA was about 16 mM, and the k(cat) of a/CaNA was 10.03 s(-1) using pNPP as substrate. With RII peptide as a substrate, the Km of a/CaNA was about 21 microM and the k(cat) of a/CaNA was 0.51 s(-1). The optimum reaction temperature was about 45 degrees C, and the optimum reaction pH was about 7.2. Our results indicate that a/CaNA is the catalytic core of CaNA, and CaN and the B-subunit binding domain itself might play roles in the negative regulation of the phosphatase activity of CaN. The results provide the basis for future studies on the catalytic domain of CaN.     

Lipoxygenase activity during parabolic flights

Experiments in Space clearly show that various cellular processes, such as growth rates, signaling pathways and gene expression, are modified when cells are placed under conditions of weightlessness. As yet, there is no coherent explanation for these observations, though recent experiments, showing that microtubule self-organization is gravity-dependent suggest that investigations at the molecular level might fill the gap between observation and understanding of Space effects. Lipoxygenases are a family of dioxygenases which have been implicated in the pathogenesis of several inflammatory conditions, in atherosclerosis, in brain aging and in HIV infection. In plants, lipoxy-genases favour germination, participate in the synthesis of traumatin and jasmonic acid and in the response to abiotic stress. Here, we took advantage of a fibre optics spectrometer developed on purpose, the EMEC (Effect of Microgravity on Enzymatic Catalysis) module, to measure the dioxygenation reaction by pure soybean lipoxygenase-1 (LOX-1) during the 28th parabolic flight campaign of the European Space Agency (ESA). The aim was to ascertain whether microgravity can affect enzyme catalysis.     

The effect of modification of sulfhydryl groups in soybean lipoxygenase-1

Soybean lipoxygenase-1 was found to contain five free sulfhydryl groups and no disulfide bridges. Three sulfhydryl groups react readily with methylmercuric halides. This modification results in significant changes of the catalytic properties of the enzyme. Comparison of modified and native lipoxygenase-1 shows the following: 1. The catalytic constant of the oxygenation of linoleic acid is reduced by approximately 50%, whereas the affinity towards linoleic acid remains unaltered. 2. At high concentrations of substrate and low concentrations of enzyme the kinetic lag phase in the oxygenation is considerably longer. 3. The regio- and stereospecificities of the oxygenation are significantly lower. 4. Besides hydroperoxides, oxo-octadecadienoic acids (4%) are formed during the oxygenation. 5. The cooxidation capacity is considerably enhanced. Treatment of methylmercury-modified lipoxygenase-1 with NaHS results in the complete recovery of the sulfhydryl groups and of the catalytic properties.     

Purification and characterization of RihC, a xanthosine-inosine-uridine-adenosine-preferring hydrolase from Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium normally salvage nucleobases and nucleosides by the action of nucleoside phosphorylases and phosphoribosyltransferases. In contrast to Escherichia coli, which catabolizes xanthosine by xanthosine phosphorylase (xapA), Salmonella cannot grow on xanthosine as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 kDa that can hydrolyze xanthosine, inosine, adenosine and uridine with similar catalytic efficiency (k(cat)/Km=1 to 4 x 10(4) M(-1)s(-1)). Cytidine and guanosine is hydrolyzed with approximately 10-fold lower efficiency (k(cat)/Km=0.7 to 1.2 x 10(3) M(-1)s(-1)) while RihC is unable to hydrolyze the deoxyribonucleosides thymidine and deoxyinosine. The Km for all nucleosides except adenosine is in the mM range. The pH optimum is different for inosine and xanthosine and the hydrolytic capacity (k(cat)/Km) is 5-fold higher for xanthosine than for inosine at pH 6.0 while they are similar at pH 7.2, indicating that RihC most likely prefers the neutral form of xanthosine.     

Characterization of D-lactate dehydrogenase producing D-3-phenyllactic acid from Pediococcus pentosaceus

D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s(-1), and 100 (mmol/L)(-1) s(-1) respectively.     

Stopped-flow kinetic investigations of the activation of soybean lipoxygenase-1 and the influence of inhibitors on the allosteric site

Herein, we report on the role of the allosteric site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The K(D) for the activation was determined to be 25.9 +/- 2.3 microM and the rate constant for the oxidation of the iron cofactor, k(2), to be 182 +/- 4 s(-1). Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a K(i) of 13.7 +/- 1.3 microM for the catalytic site and a K(i)' of 140 +/- 9 microM for the allosteric site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a K(i) of 17.5 +/- 3.8 microM. These results suggest that OS binds to an allosteric site that is separate from the catalytic iron site. We further observed that the allosteric site binding selectivity is sensitive to inhibitor length as seen by its preference for OS over that of PS, which is two carbons longer than PS.     

Kinetic investigations of the rate-limiting step in human 12- and 15-lipoxygenase

Mammalian lipoxygenases have been implicated in several inflammatory disorders; however, the details of the kinetic mechanism are still not well understood. In this paper, human platelet 12-lipoxygenase (12-hLO) and human reticulocyte 15-lipoxygenase-1 (15-hLO) were tested with arachidonic acid (AA) and linoleic acid (LA), respectively, under a variety of changing experimental conditions, such as temperature, dissolved oxygen concentration, and viscosity. The data that are presented show that 12-hLO and 15-hLO have slower rates of product release (k(cat)) than soybean lipoxygenase-1 (sLO-1), but similar or better rates of substrate capture for the fatty acid (k(cat)/K(M)) or molecular oxygen [k(cat)/K(M(O)2)]. The primary, kinetic isotope effect (KIE) for 15-hLO with LA was determined to be temperature-independent and large ((D)k(cat) = 40 +/- 8), over the range of 10-35 degrees C, indicating that C-H bond cleavage is the sole rate-limiting step and proceeds through a tunneling mechanism. The (D)k(cat)/K(M) for 15-hLO, however, was temperature-dependent, consistent with our previous results [Lewis, E. R., Johansen, E., and Holman, T. R. (1999) J. Am. Chem. Soc. 121, 1395-1396], indicating multiple rate-limiting steps. This was confirmed by a temperature-dependent, k(cat)/K(M) solvent isotope effect (SIE), which indicated a hydrogen bond rearrangement step at low temperatures, similar to that of sLO-1 [Glickman, M. H., and Klinman, J. P. (1995) Biochemistry 34, 14077-14092]. The KIE could not be determined for 12-hLO due to its inability to efficiently catalyze LA, but the k(cat)/K(M) SIE was temperature-independent, indicating distinct rate-limiting steps from both 15-hLO and sLO-1.     

Production,isolation and characterization of a sterol esterase from Ophiostoma piceae

We studied extracellular sterol esterase production by the ascomycete Ophiostoma piceae in liquid culture. Esterase activity was found in low levels in glucose medium but it was strongly induced by olive oil. An esterase was purified from the 0.5% olive oil-supplemented cultures using ultrafiltration followed by a single chromatographic step on a hydrophobic interaction column. The enzyme was a glycoprotein with 8% N-linked carbohydrate content, a molecular mass by SDS/PAGE around 56.5 kDa and an isoelectric point of 3.3. Its N-terminal sequence was TTVNVKYPEGEVV. Substrate specificity studies showed that the O. piceae esterase hydrolyzes p-nitrophenol esters, tributyrin, triolein and different cholesterol esters. Both affinity (Km) and catalytic constant (k(cat)) were positively affected by the length of the fatty acid esterifying glycerol and cholesterol. The presence of double bonds in the acyl chain increased the enzyme efficiency, although it affected the k(cat) values rather than the Km on the cholesterol esters. The O. piceae enzyme showed no interfacial activation. This enzyme could have biotechnological applications in paper manufacturing since it efficiently hydrolyzes both triglycerides and sterol esters, which form pitch deposits during manufacturing of softwood and hardwood paper pulps, respectively.     

"Critical periods" in vestibular development or adaptation of gravity sensory systems to altered gravitational conditions?

1. A feature of sensory, neuronal and motor systems is the existence of a critical period during their development. Modification of environmental conditions during this specific period of life affects development in a long-term manner, or even irreversibly. Deprivation is the prefered approach to study the existence and duration of critical periods. For gravity sensory systems, space flights offer the only opportunity for deprivation conditions. 2. Studies in a fish (Oreochromis mossambicus) and an amphibian (Xenopus laevis) revealed a significant sensitivity of their roll-induced static vestibuloocular reflex (rVOR) to a 9- to 10-day gravity deprivation (microgravity) during a spaceflight. In some instances, the rVOR was augmented after the flight as demonstrated in young Oreochromis which were launched when their rVOR had not been developed, and in Xenopus tadpoles launched after their rVOR had developed. Fish which could perform the rVOR at launch were insensitive to microgravity exposure. A similar insensitivity to microgravity was observed in Xenopus tadpoles with normal body shape which had not yet developed their rVOR at launch. Some tadpoles, however, developed an upward bended tail during their space flight; their rVOR was significantly depressed after termination of microgravity independent of the age at onset of the flight. Hypergravity depressed the rVOR for all so far tested developmental stages in both Oreochromis and Xenopus. 3. Both adaptive processes during exposure to altered gravity as well as the existence of a critical period in vestibular development might be responsible for the modulation of the rVOR recorded after exposure to altered gravity. Deprivation studies have to be extended to older developmental stages to test the possibility of a critical period; however, this approach is limited due to the low number of space flights.     

pH dependence and solvent deuterium oxide kinetic isotope effects on Bacillus cereus beta-lactamase I catalyzed reactions

The solvent kinetic isotope effects (SKIE's) on k(cat) (D(V)) and on k(cat/Km[D(V/K)] were determined for the Bacillus cereus beta-lactamase I catalyzed hydrolysis of five substrates that have values of k(cat)/K(m) varying over the range (0.014-46.3) X 10(6)M(-1) s(-1) and of k(cat) between 0.5 and 2019 s(-1). The variation of D(V/K) was only from 1.06 to 1.25 among these compounds and that in D(V) was from 1.50 to 2.16. These results require that Dk(1), the SKIE on the enzyme-substrate association rate constant, and D(k-1/k2), that on the partition ratio of the ES complex, both be near 1. The larger SKIE observed on D(V) requires that an exchangeable proton be in flight for either or both the acylation and the deacylation reaction. The pH dependence of the values k(cat)/K(m) for three substrates shows identical pK(a)s of 5.5. and 8.4. This identity combined with the fact that only one of these three substrates is kinetically "sticky" proves that the substrates can combine productively with only one protonic form of the enzyme. There is considerable substrate variation in the pK(a) values of k(cat) observed vs. pH profiles; the inflection points for all substrates studied are at pH values more extreme than are observed in the pH profiles for k(cat)/K(m).     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Differential substrate behaviour of phenol and aniline derivatives during oxidation by horseradish peroxidase: kinetic evidence for a two-step mechanism

The catalytic constant (k(cat)) and the second-order association constant of compound II with reducing substrate (k(5)) of horseradish peroxidase C (HRPC) acting on phenols and anilines have been determined from studies of the steady-state reaction velocities (V(0) vs. [S(0)]). Since k(cat)=k(2)k(6)/k(2)+k(6), and k(2) (the first-order rate constant for heterolytic cleavage of the oxygen-oxygen bond of hydrogen peroxide during compound I formation) is known, it has been possible to calculate the first-order rate constant for the transformation of each phenol or aniline by HRPC compound II (k(6)). The values of k(6) are quantitatively correlated to the sigma values (Hammett equation) and can be rationalized by an aromatic substrate oxidation mechanism in which the substrate donates an electron to the oxyferryl group in HRPC compound II, accompanied by two proton additions to the ferryl oxygen atom, one from the substrate and the other the protein or solvent. k(6) is also quantitatively correlated to the experimentally determined (13)C-NMR chemical shifts (delta(1)) and the calculated ionization potentials, E (HOMO), of the substrates. Similar dependencies were observed for k(cat) and k(5). From the kinetic analysis, the absolute values of the Michaelis constants for hydrogen peroxide and the reducing substrates (K(M)(H(2)O(2)) and K(M)(S)), respectively, were obtained.     

Reaction mechanism for mammalian pyruvate dehydrogenase using natural lipoyl domain substrates

The pyruvate dehydrogenase (E1) component of the pyruvate dehydrogenase complex (PDC) catalyzes a two-step reaction. Recombinant production of substrate amounts of the lipoyl domains of the dihydrolipoyl transacetylase (E2) component of the mammalian PDC allowed kinetic characterization of the rapid physiological reaction catalyzed by E1. Using either the N-terminal (L1) or the internal (L2) lipoyl domain of E2 as a substrate, analyses of steady state kinetic data support a ping pong mechanism. Using standard E1 preparations, Michaelis constants (Km) were 52 +/- 14 microM for L1 and 24.8 +/- 3.8 microM for pyruvate and k(cat) was 26.3 s(-1). With less common, higher activity preparations of E1, the Km values were > or =160 microM for L1 and > or =35 microM for pyruvate and k(cat) was > or =70 s(-1). Similar results were found with the L2 domain. The best synthetic lipoylated-peptide (L2 residues 163-177) was a much poorer substrate (Km > or =15 mM, k(cat) approximately equals 5 s(-1); k(cat)/Km decreased >1,500-fold) than L1 or L2, but a far better substrate in the E1 reaction than free lipoamide (k(cat)/Km increased >500-fold). Each lipoate source was an effective substrate in the dihydrolipoyl dehydrogenase (E3) reaction, but E3 had a lower Km for the L2 domain than for lipoamide or the lipoylated peptides. In contrast to measurements with slow E1 model reactions that use artificial acceptors, we confirmed that the natural E1 reaction, using lipoyl domain acceptors, was completely inhibited (>99%) by phosphorylation of E1 and the phosphorylation strongly inhibited the reverse of the second step catalyzed by E1. The mechanisms by which phosphorylation interferes with E1 activity is interpreted based on accrued results and the location of phosphorylation sites mapped onto the 3-D structure of related alpha-keto acid dehydrogenases.     

Improvement of efficiency of the Ce(IV)-induced DNA scission--relationship between the kinetic parameters (k(cat) and Km) and the DNA structure.

The Michaelis constant (Km) for double-stranded DNA, single-stranded DNA, and dinucleotide hydrolysis by Ce(IV) ion are 4.4, 15, and more than 40 mM, respectively. The order of the k(cat), however, is dinucleotide > oligonucleotides. Not only the improvement of k(cat) but also that of Km is important for the design of an efficient artificial nuclease.     

Kinetic isotope effects in the oxidation of arachidonic acid by soybean lipoxygenase-1

The reaction of soybean lipoxygenase-1 with linoleic acid has been extensively studied and displays very large kinetic isotope effects. In this work, substrate and solvent kinetic isotope effects as well as the viscosity dependence of the oxidation of arachidonic acid were investigated. The hydrogen atom abstraction step was rate-determining at all temperatures, but was partially masked by a viscosity-dependent step at ambient temperatures. The observed KIEs on k(cat) were large ( approximately 100 at 25 degrees C).     

Effect of simulated microgravity on human lymphocytes

During space flight the function of the immune system changes significantly. Several papers reported that postflight the number and the proportion of circulating leukocytes in astronauts are modified (Leach, 1992), the in vitro mitogen induced T cell activation is depressed (Cogoli et al., 1985; Konstantinova et al. 1993) and there are detectable differences in cytokine production of leukocytes as well (Talas et al. 1983; Batkai et al. 1988; Chapes et al. 1992). One of the possible modifying forces is the microgravity condition itself. Our aim was to analyse mechanisms responsible for changing leukocyte functions in low gravity environment. For terrestrial simulation of microgravity we used a Rotary Cell Culture System (RCCS) developed by NASA. We investigated the effect of simulated microgravity on separated human peripheral blood mononuclear cells (PBMCs). We detected the populations of different cells by antibodies conjugated to fluorofors using a Flow Cytometer. Since space flight reduces the number of peripheral blood lymphocytes (Stowe et al., 1999) we supposed that apoptotic (programmed cell death) processes might be involved. This hypothesis was supported by the result of our earlier experiment demonstrating that simulated microgravity increased the level of secreted Tumor Necrosis Factor-alpha (TNFalpha, a known apoptotic signal molecule) significantly (Batkai et al. 1999).     

Antibody binding in altered gravity: implications for immunosorbent assay during space flight

A single antibody-incubation step of an indirect, enzyme-linked immunosorbent assay (ELISA) was performed during microgravity, Martian gravity (0.38 G) and hypergravity (1.8 G) phases of parabolic flight, onboard the NASA KC-135 aircraft. Antibody-antigen binding occurred within 15 seconds; the level of binding did not differ between microgravity, Martian gravity and 1 G (Earth's gravity) conditions. During hypergravity and 1 G, antibody binding was directly proportional to the fluid volume (per microtiter well) used for incubation; this pattern was not observed during microgravity. These effects in microgravity may be due to "fluid spread" within the chamber (observed during microgravity with digital photography), leading to greater fluid-surface contact and subsequently antibody-antigen contact. In summary, these results demonstrate that: i) ELISA antibody-incubation and washing steps can be successfully performed by human operators during microgravity, Martian gravity and hypergravity; ii) there is no significant difference in antibody binding between microgravity, Martian gravity and 1 G conditions; and iii) a smaller fluid volume/well (and therefore less antibody) was required for a given level of binding during microgravity. These conclusions indicate that reduced gravity would not present a barrier to successful operation of immunosorbent assays during spaceflight.     

Biochemical characterization of human enteropeptidase light chain

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.