Non-competitive Inhibition Pattern Research Articles

Reaction mechanism of aspartate transcarbamylase from mouse spleen

The reaction mechanism of aspartate transcarbamylase from mouse spleen has been determined, using steady-state kinetics, isotope-exchange experiments, inhibition studies with a transition-state analog, and product-inhibition studies. Intersecting reciprocal plots obtained when one substrate was varied against different concentrations of the second substrate indicate that the mechanism is sequential. The transition-state analog, N-(phosphonacetyl)- l-aspartate, was a powerful inhibitor of aspartate transcarbamylase, with an inhibition constant ( K i ) of 2.6 × 10 −8 m at 37 °C and pH 7.4 in 0.05 m Na HEPES buffer. PALA gave competitive inhibition with carbamyl phosphate and noncompetitive inhibition with l-aspartate, indicating that carbamyl phosphate must bind before aspartate for catalysis to occur. A ping-pong mechanism in which carbamyl phosphate binds first was excluded by isotope-exchange experiments, since [ 32P]inorganic phosphate was not incorporated into carbamyl phosphate in the absence of aspartate. Product-inhibition studies showed that only inorganic phosphate and carbamyl phosphate gave a competitive pattern; all other combinations of substrate and product gave noncompetitive inhibition patterns when incubations were carried out at subsaturating concentrations of the second substrate. These inhibition patterns showed that carbamyl phosphate binds first, aspartate binds second, carbamyl aspartate dissociates first, and phosphate dissociates second.

α-Keto Acid Dehydrogenase Complexes: XX. A KINETIC STUDY OF THE PYRUVATE DEHYDROGENASE COMPLEX FROM BOVINE KIDNEY

Abstract The mammalian pyruvate dehydrogenase complex contains a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (a flavoprotein) are joined. The lipoyl moiety is bound covalently to the transacetylase and, presumably, rotates between the catalytic centers of the three different enzymes that comprise the complex. The kinetic mechanism of the pyruvate dehydrogenase complex from bovine kidney mitochondria has been investigated. Initial velocity patterns were a series of parallel lines, regardless of which substrate was varied at fixed levels of a second substrate. Product inhibition patterns showed that acetyl-CoA is competitive versus CoA and NADH is competitive versus NAD, and that both acetyl-CoA and NADH are uncompetitive versus pyruvate. These results are consistent with the patterns predicted from rate equations derived by Cleland for three-site ping-pong mechanisms. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for acetyl-CoA versus NAD and for NADH versus CoA. Evidence is presented which suggests that these anomalous product inhibition patterns are due to physical association of the flavoprotein with the transacetylase so that combination of acetyl-CoA with the transacetylase hinders combination of NAD with the flavoprotein and combination of NADH with the flavoprotein hinders combination of CoA with the transacetylase.

Enzymes in facultative anaerobiosis of molluscs-II. Basic catalytic properties of phosphoenolpyruvate carboxykinase in oyster adductor muscle

1. 1. p-Enolpyruvate carboxykinase (E.C. 4.1.1.32) from oyster adductor tissue occurs as a single activity peak with a pI value of 6·64. 2. 2. The enzyme is present in high activities only in adductor tissue. 3. 3. It exhibits a relatively high degree of specificity for IDP and GDP and is inactive in the presence of IMP, GMP and ADP. Oyster enzyme, unlike p-enolpyruvate carboxykinases from other sources, exhibits no activity in the presence of Mg 2+. 4. 4. The pH activity profiles in the presence of Zn 2+ and Mn 2+ are similar in general pattern but the pH optima are 5·1 and 6·0 respectively. 5. 5. Kinetic constants for metal ions, IDP and p-enolpyruvate were determined at different pH values. For metal ions, the apparent enzyme-Mn 2+ affinity is greater at pH 6·0 than at pH 5·1 while the apparent enzyme-Zn 2+ affinity is greater at pH 5·1 than at pH 6·0. S 0·5 values for both metal ions under different conditions vary between 0·15 and 0·52 mM. The absolute values of S 0·5 for IDP are comparable in the presence of Zn 2+ and Mn 2+ (in the range of 0·03–0.06 mM) and these are largely independent of pH. p-Enolpyruvate saturation exhibits aberrant behaviour in the present of Mn 2+ while in the presence of Zn 2+ it follows Michaelis-Menten patterns. Equally significant, the apparent K m values are at least one order of magnitude less than observed for the enzyme in the presence of Mn 2+. 6. 6. Low concentrations of Cu 2+ (10–40 μM) inhibit the enzyme in the presence of either Zn 2+ or Mn 2+. Cu 2+ in the presence of Mn 2+ exhibits a competitive inhibition pattern while in the presence of Zn 2+, a non-competitive inhibition pattern occurs. 7. 7. The physiological role of the enzyme is discussed in relation to its cellular distribution, metal ion requirements and pH properties.

Enzymes in facultative anaerobiosis of molluscs—III. Phosphoenolpyruvate carboxykinase and its role in aerobic-anaerobic transition

1. 1. In this paper, the regulatory properties of p-enolpyruvate carboxykinase (E.C. 4.1.1.32) from oyster adductor tissue are further studied. 2. 2. Competitive inhibition patterns are obtained for metal ions with ITP as the inhibitor. In both instances 0·25 mM ITP increases the apparent K m values of the metal ion by tenfold. 3. 3. In the presence of Zn 2+ with ITP as the inhibitor and IDP as the variable substrate, a linear non-competitive inhibition pattern is obtained. At all concentrations of ITP tested the K m (IDP) remains unchanged at about 0·06 mM. In contrast in the presence of Mn 2+ a linear, mixed competitive ITP inhibition pattern is obtained. In this instance, 0·25 mM ITP increases the K m (IDP) several fold. 4. 4. In the presence of either metal ion, ITP inhibition is competitive with respect to p-enolpyruvate. With Zn 2+, 0·5 mM ITP increases the t m (p-enolpyruvate) by about twofold while in the presence of Mn 2+, 0·25 mM ITP increases the K m (p-enolpyruvate) at least by fivefold. 5. 5. In the presence of IDP or GDP, GTP also inhibits the p-enolpyruvate carboxykinase reaction. 6. 6. Alanine has a slight stimulatory effect at low p-enolpyruvate concentrations and markedly deinhibits ITP (or GTP) inhibited p-enolpyruvate carboxykinase. 7. 7. From the data presented it is speculated that co-ordinated changes in the intracellular concentrations of H +, ITP, GTP and alanine control p-enolpyruvate carboxykinase activity in vivo.

The effect of SO 3 -- on the activity of ribulose-1,5-diphosphate carboxylase in isolated spinach chloroplasts

SO 3 (--) inhibits the activity of ribulose-1,5-diphosphate carboxylase in isolated spinach chloroplasts. It shows a non-competitive inhibition pattern with respect to ribulose-1,5-diphosphate and Mg(++) but a competitive one with respect to HCO 3 (-) . The K i -values are 14 mM SO 3 (--) and 9.5 mM SO 3 (-) respectively for the non-competitive inhibition but only 3.0 mM SO 3 (--) in the case of competitive inhibition with HCO 3 (--) as a substrate. Thus it is concluded that the competitive inhibition type will predominate at low SO 3 (--) and low internal CO2 concentrations.

Metal Toxicity to Sewage Organisms

The Michaelis-Menten model of enzyme inhibition is a convenient method to measure toxicity and to give insights to toxic behavior in pure culture and simple substrate conditions. A linear noncompetitive inhibition pattern is demonstrated for sewage bacteria and a linear competitive inhibition pattern for sewage fungus Geotrichum candidum. Silver and nickel are more toxic to sewage bacteria than to Geotrichum candidum and therefore the metal toxicity is believed to be connected with the overgrowth of the fungus in activated sludge processes.

3,4-Dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione 4,5-Dioxygenase from Nocardia restrictus: II. KINETIC STUDIES

Abstract Kinetic studies with 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione-4,5-dioxygenase gave intersecting initial velocity plots that conform to a sequential mechanism. Linear noncompetitive product inhibition patterns were observed with respect to either substrate, indicating the formation of a dead end complex. 4-Isopropyl catechol, a structural analogue of the organic substrate, inhibited the enzyme competitively with respect to the organic substrate as expected, but inhibited the enzyme uncompetitively with respect to molecular oxygen. These results are consistent with an ordered Bi-Uni mechanism where molecular oxygen is added first, followed by organic substrate, and the product is then released. Detailed analysis of the kinetic data suggests the existence of a dead end enzyme-oxygen-product complex.

Relation between conformations and activities of lipoamide dehydrogenase. IV. Apoenzyme structure and flavin binding aspects

1. 1. The apoenzyme of lipoamide dehydrogenase (NADH: lipoamide oxido-reductase, EC 1.6.4.3) is thermolabile and urea-sensitive; the stability is also concentration-dependent. Upon prolonged incubation on ice the tertiary structure of concentrated apoenzyme (1 mg/ml) changes, though the flavin binding site is less affected. The apoenzyme partially withstands freezing for several weeks. 2. 2. Compared with that of the holoenzyme the protein fluorescence is increased in the apoenzyme; the excitation spectrum shows two maxima at 284 nm and 290 nm. Resolution of the difference fluorescence emission spectra showed a tyrosine contribution in the 305-nm region and non-polar tryptophan emission around 330 nm. 3. 3. The return of the lipoate activity is dependent on the apoenzyme concentration. Recombination systems with high apoenzyme concentrations (0.8 mg/ml) and excess of flavin develop a higher lipoate activity whilst the DCIP activity decreases faster than in the case of at lower apoenzyme concentrations (0.1 mg/ml). The dimerization is dependent on pH (7.2–7.5) and ionic strength (0.2 M). Urea, p- chloromercuribenzoate (PCMB) and l-cysteine disturb the recombination. 4. 4. Flavin derivatives are able to bind with the apoenzyme. Flavin-8-bromoadenine dinucleotide (FBAD) partially restores the DCIP activity ( K ass = 1.5·10 4 M −1 ). FMN is a competitive inhibitor of FAD binding and does not restore any activity. FMN derivatives generally show a non-competitive inhibition pattern, with respect to restoration of the DCIP activity. 3(N)-adducts of FMN exert very little inhibition. 2-Derivatives, such as 2-thio-FMN and 2-NH-phenyl-FMN, are less effectively bound, as also is tetra-hydro-FMN. The inhibitors induce, like FAD, conformational changes in the protein, resulting in a time-dependent increase of the binding forces between apoenzyme and flavin. 5. 5. Parts other than the isoalloxazine moiety of the flavin molecule, viz. the adenine part and pyrophosphate, are also involved in the FAD binding: NADH, NAD +, ADP, adenine, ATP and pyrophosphate inhibit the flavin binding. All nucleotides which inhibit the FAD binding affect the dimerization; only NAD + has a different behaviour in this respect since it promotes the return of the lipoate activity.

Initial Rate Studies of Adenylosuccinate Synthetase with Product and Competitive Inhibitors

The kinetic mechanism of action of adenylosuccinate synthetase from Escherichia coli, strain B, was investigated from initial rate studies. The kinetic data are consistent with a sequential mechanism for the enzyme. A rapid equilibrium, fully random mechanism was found to be in harmony with the kinetic findings from studies with competitive and product inhibitors. This result serves to explain why partial exchange reactions are not observed with adenylosuccinate synthetase. It is not, however, possible to ascertain the nature of the catalytic transition state. It was observed that competitive inhibitors for a single substrate acted as noncompetitive inhibitors relative to the other substrates. Similarly, guanosine diphosphate functions as a competitive product inhibitor of GTP, but shows noncompetitive inhibition patterns for inosine monophosphate and aspartate. Adenylosuccinate was found to be competitive with respect to IMP and noncompetitive relative to the other substrates. A procedure is presented for replotting initial rate data for three-substrate systems which permits a choice of mechanism to be made from among sequential mechanisms.

Steady-state kinetics of one-substrate enzymic mechanisms involving two enzyme conformations I. Effects of modifiers on a mechanism postulating a single enzyme-substrate complex

Steady-state kinetics have been worked out for a one-substrate enzymic mechanism involving two interconvertible conformations of the enzyme protein: one able to bind the substrate and the other regenerated by the reaction. Effects of modifiers, assumed to bind to both enzyme conformations with exclusion of the substrate, have been considered. It has been shown that such modifiers may give uncompetitive or non-competitive inhibition patterns. If the two enzyme-modifier complexes, formed by a given modifier, are assumed to be interconvertible, uncompetitive activation is possible. Such a modifier can also remove the uncompetitive inhibition produced by another modifier.

The role of sulphydryl groups in soybean beta-amylase.

Summary This research was undertaken to explore the possible role of sulphydryl groups in the activity of soybean β -amylase ( α -1,4 glucan maltohydrolase, EC 3.2.1.2). The dependence of initial velocity and the independence of K m on pH, as well as the non-competitive inhibition pattern of soybean β -amylase by p -mercuribenzoate (PMB), indicate that sulphydryl groups do not participate in binding the substrate and their role in enzymatic action is related to the catalytic step only. The extent of inhibition of soybean β -amylase by PMB is inversely related to the concentration of the acetate buffer used, indicating the “protecting” effect of acetate ions. “Competitive-like” inhibition of β -amylase by PMB was achieved when the concentration of acetate buffer used in the assay was changed in parallel with the dilution of the substrate. Similar results were obtained with crystalline sweet potato β -amylase. Partial titration of soybean β -amylase by PMB inhibited the activity to different extents: titration of one of the five SH groups present in one enzyme molecule affects activity only very slightly, whereas titration of all the 5 SH groups results in almost complete loss of activity.