Enzymatic Substrate Inhibition in Metal Free Catecholase Activity

The catecholase activities were routinely modeled using transition metal complexes as catalyst and in some case basic pH were used as a reaction condition. In this article, the catalytic aerobic oxidation of proxy substrate 3,5-di-tert-butylcatechol (DTBC) in methanol using triethylamine/diethylamine as catalyst was demonstrated as a functional mimic of catecholase activity. The kinetic manifestation of DTBC oxidation was explained as enzymatic substrate inhibition pattern in Michaelis-Menten kinetic model. The mechanistic insight of the aerobic oxidation of DTBC was further validated using various spectroscopic techniques and DFT methods.     

Inhibition of Proteolysis by a Cyclooxygenase Inhibitor,Indomethacin

The effect of indomethacin, a non-steroidal anti-inflammatory drug upon purified calpain has been studied. Also, its effects upon Ca²⁺-mediated degradation of cytoskeletal proteins (neurofilament) in spinal cord homogenate has been investigated. A dose-dependent inhibition of purified calpain activity was observed. A 50% inhibition of ¹⁴C-caseinolytic activity was obtained with less than 1.1 mM of indomethacin while the activity was completely inhibited at 3.3 mM concentration. The inhibitory effect of ketorlac, another non-steroidal anti-inflammatory drug, upon calpain was weaker than that of indomethacin. The degradation of myelin basic protein (MBP) by cathepsin B, a lysosomal cysteine protease, was significantly inhibited by indomethacin. It also inhibited the Ca²⁺-mediated degradation of neurofilament protein (NFP) in spinal cord homogenate. The extent of NFP degradation was analyzed by SDS-PAGE and the inhibition shown by indomethacin was weaker than that observed with leupeptin and the calpain inhibitor E64-d. The inhibitory effect of indomethacin on the activity of multicatalytic proteinase complex was negligible. These results suggest that indomethacin, a non-steroidal anti-inflammatory drug and cyclooxygenase inhibitor also inhibits proteinases, including cathepsin B and calpain.     

胰岛素受体底物活性调控的分子机制

胰岛素受体底物(insulin receptor substrate,IRS)是胰岛素信号转导通路中一个极其重要的信号分子,对胰岛素信号级联效应具有至关重要的作用。目前有关胰岛素受体底物活性调节的研究主要集中在两个方面,一方面是磷酸化水平的调节机制,另一方面是细胞因子信号阻抑剂(suppressor of cytokine signaling,COCS)所介导的直接和间接调控。了解胰岛素受体底物活性调节机制将有助于进一步探索胰岛素抵抗和Ⅱ型糖尿病的发病机制。     

Substrate Inhibition of Transglucosyl-Amylase by Maltose

Transglucosyl-amylase was inhibited by maltose when maltose served as a substrate. As a function of substrate concentration, the rates initially rose proportionately with increases of maltose levels until a maximal rate was attained. Further increases of maltose concentration decreased reaction rates. The attainment of a maximal rate with increasing substrate concentration and close correspondence between experimental and calculated rates indicated the involvement of ternary complex formation. Evidence also suggested that substrate inhibition is caused by maltose competing with water for the acceptor sites during complex formation.     

The Molecular Mechanism of Substrate Engagement and Immunosuppressant Inhibition of Calcineurin

Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca²⁺/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, “LxVP.” The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that “LxVP” sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent “LxVP”-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.     

双底物酶促反应的底物抑制分析

存在于双底物随机机制的途径中,偏离米氏方程行为的底物抑制现象被作为一个问题进行探讨．通过计算机模拟出双底物浓度和稳态下的初始速率构成的三维图形,提出底物抑制、速率常数和底物浓度之间的关系．     

Urease Inhibition by Polymeric Substrate Analogues and Thiophosphamides

Inhibition of soybean urease by polymeric substrate analogues, urea and thiourea polydisulfides (PDSU and PDSTU, respectively); or three thiophosphoric acid amides (TPAA), tri-(N-3-hydroxyphenyl)thiophosphamide (1), tri-(N-4,4"-aminodiphenyl)thiophosphamide, and di-oxy-(N--piridyl)thiophosphamide (3) was studied in aqueous solutions at various pH values. The inhibitory effects of all these substances were reversible and competitive with the lowest inhibition constant K _i2.8 M for TPAA-1 at pH 3.85. Above and below this pH value, K _iincreased, reaching 24 M at pH 7.2. All test substances inhibited urease comparably with known inhibitors such as thiols (cysteamine, etc.) and hydroxamic acid derivatives, but were less efficient than phosphorodiamidates. Structural features of possible urease inhibitors of higher efficiency were proposed.     

Consistent Inhibition of Cyclooxygenase Drives Macrophages towards the Inflammatory Phenotype

Macrophages play important roles in defense against infection, as well as in homeostasis maintenance. Thus alterations of macrophage function can have unexpected pathological results. Cyclooxygenase (COX) inhibitors are widely used to relieve pain, but the effects of long-term usage on macrophage function remain to be elucidated. Using bone marrow-derived macrophage culture and long-term COX inhibitor treatments in BALB/c mice and zebrafish, we showed that chronic COX inhibition drives macrophages into an inflammatory state. Macrophages differentiated in the presence of SC-560 (COX-1 inhibitor), NS-398 (COX-2 inhibitor) or indomethacin (COX-1/2 inhibitor) for 7 days produced more TNFα or IL-12p70 with enhanced p65/IκB phosphoylation. YmI and IRF4 expression was reduced significantly, indicative of a more inflammatory phenotype. We further observed that indomethacin or NS-398 delivery accelerated zebrafish death rates during LPS induced sepsis. When COX inhibitors were released over 30 days from an osmotic pump implant in mice, macrophages from peritoneal cavities and adipose tissue produced more TNFα in both the basal state and under LPS stimulation. Consequently, indomethacin-exposed mice showed accelerated systemic inflammation after LPS injection. Our findings suggest that macrophages exhibit a more inflammatory phenotype when COX activities are chronically inhibited.     

Induction of Superoxide Dismutase by Molecular Oxygen

Oxygen induces superoxide dismutase in Streptococcus faecalis and in Escherichia coli B. S. faecalis grown under 20 atm of O(2) had 16 times more of this enzyme than did anaerobically grown cells. In the case of E. coli, changing the conditions of growth from anaerobic to 5 atm of O(2) caused a 25-fold increase in the level of superoxide dismutase. Induction of this enzyme was a response to O(2) rather than to pressure, since 20 atm of N(2) was without effect. Induction of superoxide dismutase was a rapid process, and half of the maximal level was reached within 90 min after N(2)-grown cells of S. faecalis were exposed to 20 atm of O(2) at 37 C. S. faecalis did not contain perceptible levels of catalase under any of the growth conditions investigated by Stanier, Doudoroff, and Adelberg (23), and the concentration of catalase in E. coli was not affected by the presence of O(2) during growth. S. faecalis, which had been grown under 100% O(2) and which therefore contained an elevated level of superoxide dismutase, was more resistant of 46 atm of O(2) than were cells which had been grown under N(2). E. coli grown under N(2) contained as much superoxide dismutase as did S. faecalis grown under 1 atm of O(2). The E. coli which had been grown under N(2) was as resistant to the deleterious effects of 50 atm of O(2) as was S. faecalis which had been grown under 1 atm of O(2). These results are consistent with the proposal that the peroxide radical is an important agent of the toxicity of oxygen and that superoxide dismutase may be a component of the systems which have been evolved to deal with this potential toxicity.     

omega]-Hydroxylation of Oleic Acid in Vicia sativa Microsomes (Inhibition by Substrate Analogs and Inactivation by Terminal Acetylenes)

Oleic acid (18:1) is hydroxylated exclusively on the terminal methyl by a microsomal cytochrome P-450-dependent system ([omega]-OAH) from clofibrate-induced Vicia sativa L. (var minor) seedlings (F. Pinot, J.-P. Salaun, H. Bosch, A. Lesot, C. Mioskowski, F. Durst [1992] Biochem Biophys Res Commun 184: 183-193). This reaction was inactivated by two terminal acetylenes: (Z)-9-octadecen-17-ynoic acid (17-ODCYA) and the corresponding epoxide, (Z)-9,10-epoxyoctadecan-17-ynoic acid (17-EODCYA). Inactivation was mechanism-based, with an apparent binding constant of 21 and 32 [mu]M and half-lives of 16 and 19 min for 17-ODCYA and 17-EODCYA, respectively. We have investigated the participation of one or more [omega]-hydroxylase isoforms in the oxidation of fatty acids in this plant system. Lauric acid (12:0) is [omega]-hydroxylated by the cytochrome P-450 [omega]-hydroxylase [omega]-LAH (J.-P. Salaun, A. Simon, F. Durst [1986] Lipids 21: 776-779). Half-lives of [omega]-OAH and [omega]-LAH in the presence of 40 [mu]M 17-ODCYA were 23 and 41 min, respectively. Inhibition of oleic acid [omega]-hydroxylation was competitive with linoleic acid (18:2), but noncompetitive with lauric acid (12:0). In contrast, oleic acid did not inhibit [omega]-hydroxylation of lauric acid. Furthermore, 1-pentadecyltriazole inhibited [omega]-hydroxylation of oleic acid but not of lauric acid. These results suggest that distinct monooxygenases catalyze [omega]-hydroxylation of medium- and long-chain fatty acids in V. sativa microsomes.     

Inhibition of the Tyrosinase Oxidation of One Substrate by Another

The catalytic oxidation of catechol by crude preparations of mushroom tyrosinase was studied by a method yielding data on initial reaction velocities. Graphical analysis of the results suggests that an excess of catechol inhibits its own oxidation by a competitive process, thus accounting for the observed optimum in the substrate concentration. However, added phenol, though itself a substrate, inhibits the enzymatic oxidation of catechol by a process that is neither competitive nor non-competitive, but a mixture of the two types. Mechanisms of this inhibition of the enzyme by a second substrate are discussed in exploring the problem of substrate-substrate inhibition.     

Repression of Acetolactate Synthase Activity through Antisense Inhibition (Molecular and Biochemical Analysis of Transgenic Potato (Solanum tuberosum L. cv Desiree) Plants)

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, valine, and isoleucine, is the biochemical target of different herbicides. To investigate the effects of repression of ALS activity through antisense gene expression we cloned an ALS gene from potato (Solanum tuberosum L. cv Desiree), constructed a chimeric antisense gene under control of the cauliflower mosaic virus 35S promoter, and created transgenic potato plants through Agrobacterium tumefaciens-mediated gene transfer. Two regenerants revealed severe growth retardation and strong phenotypical effects resembling those caused by ALS-inhibiting herbicides. Antisense gene expression decreased the steady-state level of ALS mRNA in these plants and induced a corresponding decrease in ALS activity of up to 85%. This reduction was sufficient to generate plants almost inviable without amino acid supplementation. In both ALS antisense and herbicide-treated plants, we could exclude accumulation of 2-oxobutyrate and/or 2-aminobutyrate as the reason for the observed deleterious effects, but we detected elevated levels of free amino acids and imbalances in their relative proportions. Thus, antisense inhibition of ALS generated an in vivo model of herbicide action. Furthermore, expression of antisense RNA to the enzyme of interest provides a general method for validation of potential herbicide targets.     

Substrate Binding Sites of Leuconostoc Dextransucrase Evaluated by Inhibition Kinetics

Graphical analysis of inhibition kinetics for dextransucrase from Leuconostoc mesenteroides was done with typical inhibitors, competitive and noncompetitive. Based on the plots of Yonetani-Theorell and Semenza-Balthazar, mutual competition between the pairs of inhibitors of identical kinetic type was observed, while combination of competitive and noncompetitive inhibitors gave no significant mutual interactions. By the procedure of Nitta et al., binding sites for competitive and noncompetitive inhibitors were shown to be distant from each other. Moreover, two noncompetitive inhibitors competed with each other for a single binding site on the enzyme. Although biphasic reciprocal plots may suggest rather complicated binding of various inhibitors, the results obtained by the three graphical methods are fully explained when competitive and noncompetitive inhibitors for substrate sucrose bind to the so-called donor- and acceptor-sites of dextransucrase, respectively.     

Inhibition of Photosynthetic Oxygen Evolution in Thylakoids by Cyanide

Cyanide inhibition of oxygen evolution from isolated chloroplastswas accomplished by altering the thylakoid membrane properties(in addition to charge-screening) by high concentrations ofMg²⁺. The divalent cation facilitated the approach of the cyanideligand (pK = 9.4) to the water-splitting site. The additionof the divalent cation ionophore, A23187 [GenBank] , in the presence ofMg²⁺ enhanced the expression of cyanide inhibition of photosystemII-mediated O₂-evolution. Furthermore, electron transport partialreactions allowed the identification of the site of cyanideaction as being between the site of Tris-inhibition of electrontransport and the site of diphenylcarbazide donation on theoxidizing side of PS II. (Received October 25, 1982; Accepted December 29, 1982)     

Inhibition of the Activity of NAD(P)H-Dependent Oxidase in Pistils of Lilium longiflorum by cAMP

The effects of exogenous cAMP on the activities of the stressenzymes were studied using the extracts from stigmas and stylesof Lilium longiflorum cv. Hinomoto without pollination in relationto self-incompatibility. The activity of NADH- and NADPH-dependentoxidases (