Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism

The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated. Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released. For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP. The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive. Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate. For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P. The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P. Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP. These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes. In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released. For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.     

Inhibition and kinetics of mycobacterium tuberculosis and mycobacterium smegmatis mycothiol-S-conjugate amidase by natural product inhibitors

The current rise in mycobacterial-related infections and disease, coupled with drug resistance, underlines the continuing need for new antimycobacterials. To this end, we have screened approximately 1500 extracts derived from marine plants and invertebrates and terrestrial fungi for their ability to inhibit a newly described mycobacterial detoxification enzyme mycothiol-S-conjugate amidase (MCA). As described in this paper, our screening and chemistry efforts thus far have led to the identification of 13 natural product inhibitors that represent six different structural classes. By conducting enzyme inhibition assays using varied inhibitor and substrate concentrations, we have determined the mode of inhibition of Mycobacterium tuberculosis MCA for four of these compounds. We show that two types of bromotyrosine-derived natural products are competitive inhibitors of MCA; while oceanapiside, an alpha,omega-bis-aminohydroxy glycosphingolipid, and the fungal metabolite gliotoxin, a dithiadiketopiperazine, are simple and mixed non-competitive inhibitors, respectively. Correlation of these results with the chemical structures suggests that MCA is a metalloenzyme and that the oximinoamide and spiro-isoxazoline amide groups present in the competitive inhibitors are substrate mimics.     

UDP glucose-4 epimerase from Saccharomyces fragilis: interaction with sugar phosphates at an effector site

UDP glucose-4 epimerase from $\text{Saccharomyces fragilis}$ was found to be activated at low substrate concentrations by some metabolically related sugar phosphates. The stimulation of the enzyme activity showed a sigmoidal response to the increasing concentration of glucose-6 phosphate at a fixed substrate concentration. The activated enzyme was allosterically inhibited by UMP which otherwise acted as a strictly competitive inhibitor for the enzyme. The interaction with sugar phosphates was not accompanied by any change in the aggregation state of the molelcule.     

Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of (14)CO(2) from [1-(14)C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K(i) of 100mum. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

Human deoxycytidine kinase: kinetic mechanism and end product regulation

The kinetic properties of the monomeric deoxycytidine kinase (EC 2.7.1.74) from leukemic human T-lymphoblasts have been investigated. The results of steady-state initial-rate kinetic analysis and product inhibition studies at pH 7.5 and 37 degrees C indicate that substrate binding follows an ordered sequential pathway, with the magnesium salt of ATP being the first substrate to bind and dCMP the last product to dissociate. At subsaturating substrate concentrations, dCMP produced competitive inhibition against ATP, while against varied deoxycytidine concentrations dCMP exhibited mixed-type inhibition. ADP produced noncompetitive inhibition against either substrate. The limiting Km values for deoxycytidine and MgATP were 0.94 and 30 microM, respectively. The end product inhibitor dCTP exhibited competitive inhibition against varied ATP concentration, with a dissociation constant estimated to be 0.7 microM when extrapolated to zero ATP concentration. dCTP was purely noncompetitive against varied deoxycytidine concentration. On the basis of these kinetic results, and on the strong and specific inhibition by dCTP, it is proposed that this end product functions as a multisubstrate analogue, with its triphosphate group binding to the phosphate donor site of the enzyme and its deoxycytidine moiety overlapping and binding to the deoxynucleoside site in a highly specific manner.     

Inhibition of homogeneous human eosinophil arylsulfatase B by sulfidopeptide leukotrienes

Arylsulfatase B, purified to homogeneity from human eosinophils, is a tetrameric enzyme whose activity varied in accordance with the state of association of its monomeric subunits. The rate of dissociation of oligomeric forms was slow relative to the rate of the enzymatic reaction so that the kinetic properties of the enzyme depended on the concentration of the enzyme before assay. For concentrated enzyme solutions (14 micrograms/ml), Lineweaver-Burk analysis demonstrated substrate inhibition at greater than or equal to 20 mM substrate and revealed two distinct regions of activity at low and intermediate substrate concentrations. The addition of bovine serum albumin (60 micrograms/ml) or sucrose (0.25 M), which prevent subunit dissociation, yielded a linear relationship on Lineweaver-Burk analysis at non-inhibitory substrate concentrations. For dilute enzyme concentrations (4.7 micrograms/ml), inhibition occurred at greater than or equal to 2 mM substrate. Nanomolar amounts of leukotriene C4 (LTC4), relative to millimolar concentrations of substrate, inhibited eosinophil arylsulfatase B. On Lineweaver-Burk analysis, the pattern of inhibition of LTC4 with concentrated enzyme was compatible with competitive inhibition of only one oligomeric form of the enzyme, whereas at low enzyme concentrations the pattern of inhibition was apparently competitive. These findings suggest that LTC4 is a potent competitive inhibitor of a dissociated, possibly dimeric, form of the enzyme. Nanomolar concentrations of LTC4, leukotriene D4, and leukotriene E4 were equally inhibitory, whereas leukotriene B4 and isomeric 5,12-dihydroxyeicosatetraenoic acids had no inhibitory activity, indicating a requirement for a thiopeptide at C-6. Thiopeptide leukotriene analogs without an intact triene structure also lacked inhibitory activity. Sulfoxide analogs of LTC4 and leukotriene D4 were potent inhibitors, although two sulfone analogs of leukotriene D4 were not inhibitory. Arylsulfatase B did not inactivate the spasmogenic activity of sulfidopeptide leukotrienes. These findings indicate that sulfidopeptide leukotrienes and their sulfoxide derivatives may regulate by competitive inhibition the activity of oligomeric forms of the eosinophil lysosomal hydrolase, arylsulfatase B.     

Steady-state kinetic mechanism of rat tyrosine hydroxylase.

The steady-state kinetic mechanism for rat tyrosine hydroxylase has been determined by using recombinant enzyme expressed in insect tissue culture cells. Variation of any two of the three substrates, tyrosine, 6-methyltetrahydropterin, and oxygen, together at nonsaturating concentrations of the third gives a pattern of intersecting lines in a double-reciprocal plot. Varying tyrosine and oxygen together results in a rapid equilibrium pattern, while the other substrate pairs both fit a sequential mechanism. When tyrosine and 6-methyltetrahydropterin are varied at a fixed ratio at different oxygen concentrations, the intercept replot is linear and the slope replot is nonlinear with a zero intercept, consistent with rapid equilibrium binding of oxygen. All the replots when oxygen is varied in a fixed ratio with either tyrosine or 6-methyltetrahydropterin are nonlinear with finite intercepts. 6-Methyl-7,8-dihydropterin and norepinephrine are competitive inhibitors versus 6-methyltetrahydropterin and noncompetitive inhibitors versus tyrosine. 3-Iodotyrosine, a competitive inhibitor versus tyrosine, shows uncompetitive inhibition versus 6-methyltetrahydropterin. At high concentrations, tyrosine is a competitive inhibitor versus 6-methyltetrahydropterin. These results are consistent with an ordered kinetic mechanism with the order of binding being 6-methyltetrahydropterin, oxygen, and tyrosine and with formation of a dead-end enzyme-tyrosine complex. There is no significant primary kinetic isotope effect on the V/K values or on the Vmax value with [3,5-2H2]tyrosine as substrate. No burst of dihydroxyphenylalanine production is seen during the first turnover. These results rule out product release and carbon-hydrogen bond cleavage as rate-limiting steps.     

A simple mechanism decreasing free metabolite pool size in static spatial channelling

We propose a simple mechanism which enables decrease of the free pool of channelled metabolite in static spatial channelling, when the concentration of the enzyme consuming the channelled metabolite is greater than the concentration of the enzyme producing this metabolite. Spatial channelling occurs between two enzymes when the common metabolite is released to a small space between these enzymes and does not form a ternary covalent complex with them, as is the case in covalent (dynamic or static) channelling. The mechanism proposed is qualitatively independent of rate constants, metabolite concentrations as well as other kinetic properties and is quantitatively significant for all physiologically relevant conditions. Calculations show that the free metabolite pool must decrease, when the concentration of the enzyme consuming the channelled metabolite is greater than the enzyme producing it. This mechanism is much more effective than increase in the concentration (or rate constant) of the enzyme consuming the metabolite in the absence of spatial channelling.     

Effects of halides and bicarbonate on chloride transport in human red blood cells

Chloride self-exchange was determined by measuring the rate of 36Cl efflux from human red blood cells at pH 7.2 (0 degrees C) in the presence of fluoride, bromide, iodide, and bicarbonate. The chloride concentration was varied between 10--400 mM and the concentration of other halides and bicarbonate between 10--300 mM. Chloride equilibrium flux showed saturation kinetics. The half-saturation constant increased and the maximum flux decreased in the presence of halides and bicarbonate: the inhibition kinetics were both competitive and noncompetitive. The competitive and the noncompetitive effects increased proportionately in the sequence: fluoride less than bromide less than iodide. The inhibitory action of bicarbonate was predominantly competitive. The noncompetitive effect of chloride (chloride self-inhibition) on chloride transport was less dominant at high inhibitor concentrations. Similarly, the noncompetitive action of the inhibitors was less dominant at high chloride concentrations. The results can be described by a carrier model with two anion binding sites: a transport site, and a second site which modifies the maximum transport rate. Binding to both types of sites increases proportionately in the sequence: fluoride less than chloride less than bromide less than iodide.     

Basis for the anomalous effect of competitive inhibitors on the kinetics of hydrolysis of short-chain phosphatidylcholines by phospholipase A2.

The effect of four specific competitive inhibitors on the kinetics of hydrolysis of short-chain diacyl-sn-glycero-3-phosphocholines below their critical micelle concentrations was examined. The kinetics of hydrolysis of short-chain substrates dispersed as solitary monomers were generally consistent with the classical Michaelis-Menten formalism; i.e., hydrolysis began without any latency period, the steady-state rate was observed at higher substrate concentrations, the steady-state initial rate showed a linear dependence on the enzyme concentration, and the hyperbolic dependence of the initial rate on the substrate concentration could be described in terms of KM and Vmax parameters. The competitive nature of the inhibitors used in this study has been established by a variety of techniques, and the equilibrium dissociation constants for the inhibitors bound to the enzyme were measured by the protection method [Jain et al. (1991) Biochemistry 30, 7306-7317]. The kinetics of hydrolysis in the presence of competitive inhibitors could be described by a single dissociation constant. However, the value of the dissociation constant obtained under the kinetic conditions was comparable to that obtained by the protection method for the inhibitor-enzyme complex bound to a neutral diluent, rather than to the value of the dissociation constant obtained with solitary monomeric inhibitors and the enzyme in the aqueous phase. Spectroscopic methods showed that the effectively lower dissociation constant of an inhibitor bound to PLA2 at the interface is due to the stabilization of the enzyme-inhibitor complex by interaction with other amphiphiles present in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic effect of fructose 2,6-bisphosphate on the liver fructose-1,6-bisphosphatase: mechanistic and physiological implications

Fructose 2,6-bisphosphate has been claimed to be both a substrate analogue and an allosteric inhibitor of fructose-1,6-bisphosphatase. The results reported here show that fructose 2,6-bisphosphate can be both an inhibitor and an activator of the enzyme, depending on the substrate concentration. This biphasic behaviour at saturating concentrations of substrate can only be due to an allosteric effect. In addition to the mechanistic implication it is possible that this finding may have physiological meaning.     

Effects of acyclovir and its metabolites on purine nucleoside phosphorylase

Acyclovir (9-(2-hydroxyethoxymethyl)guanine), the clinically useful antiherpetic agent, is an "acyclic" analogue of 2'-deoxyguanosine. Purine nucleoside phosphorylase partially purified from human erythrocytes did not catalyze detectable phosphorolysis of this drug or any of its metabolites (less than 0.07% of the rate with Guo). However, these compounds were competitive inhibitors of this enzyme with Ino as the variable substrate. Acyclovir per se was a relatively weak inhibitor. Its Ki value (91 microM) was much greater than that for its 8-hydroxy metabolite (Ki = 4.7 microM) but less than that for its carboxylic acid metabolite (9-carboxymethoxy-methylguanine) (K'i = 960 microM). The phosphorylated metabolites of acyclovir were more potent inhibitors than were their guanine nucleotide counterparts. At a phosphate concentration of 50 mM, the apparent Ki values for the mono- (120 microM), di- (0.51 microM), and tri (43 microM)-phosphate esters of acyclovir were 1/2, 1/1200, and 1/26 those for dGMP, dGDP, and dGTP, respectively. The concentration of phosphate did not markedly affect the Ki value of acyclovir but dramatically affected those of its phosphorylated metabolites and their nucleotide counterparts. Decreasing phosphate to a physiological concentration (1 mM) decreased the apparent Ki values for the mono-, di-, and triphosphate esters of acyclovir to 6.6, 0.0087, and 0.31 microM, respectively. Inhibition of the enzyme by acyclovir diphosphate was also influenced by pH. This metabolite of acyclovir is the most potent inhibitor of purine nucleoside phosphorylase reported to date. It has some features of a "multisubstrate" analogue inhibitor.     

'Slave' metabolites and enzymes. A rapid way of delineating metabolic control.

Although control of fluxes and concentrations tends to be distributed rather than confined to a single rate-limiting enzyme, the extent of control can differ widely between enzymes in a metabolic network. In some cases, there are enzymes that lack control completely. This paper identifies one surprising origin of such lack of control: If, in a metabolic system, there is a metabolite that affects the catalytic rate of only one enzyme, the corresponding enzyme cannot control any metabolic variable other than the concentration of that metabolite. We call such enzymes 'slave enzymes', and the corresponding metabolites 'slave metabolites'. Implications of the existence of slave enzymes for the control properties of enzymes further down the metabolic pathway are discussed and examined for the glycolytic pathway of yeast. Inadvertent assumptions in metabolic models may cause the latter incorrectly to calculate absence of metabolic control. The phenomenon of slave enzymes may well be important in enhancing metabolic signal transduction.     

An investigation on acarbose inhibition and the number of active sites in an amylopullulanase (L14-APU) from an Iranian Bacillus sp.

An amylopullulanase (L₁₄-APU) from an Iranian thermophilic bacterium was purified and the effect of acarbose, as a general inhibitor of α-amylases, on pullulan and starch hydrolysis catalyzed by L₁₄-APU was investigated. The inhibition is a competitive type whereas inhibition constants for pullulan and starch are 99 μM and 72 μM, respectively. Investigation of the reaction rate in a system contains competitive substrates and the inhibition type of acarbose in presence of different substrates suggests that L₁₄-APU possesses only one active site for two activities. The analysis of metal ions and other reagents effects has shown that Ca²⁺, Mg²⁺, Mn²⁺ and Co²⁺ enhanced both activities of the enzyme while N-bromosuccinimide treatment leads to the complete inactivation of the enzyme. The enzyme activity increased in the presence of low concentration of SDS as a surfactant.     

Mechanistic and kinetic studies of inhibition of enzymes

A graphical method for analyzing enzyme data to obtain kinetic parameters, and to identify the types of inhibition and the enzyme mechanisms, is described. The method consists of plotting experimental data as nu/(V0 - nu) vs 1/(I) at different substrate concentrations. I is the inhibitor concentration; V0 and nu are the rates of enzyme reaction attained by the system in the presence of a fixed amount of substrate, and in the absence and presence of inhibitor, respectively. Complete inhibition gives straight lines that go through the origin; partial inhibition gives straight lines that converge on the 1-I axis, at a point away from the origin. For competitive inhibition, the slopes of the lines increase with increasing-substrate concentration; with noncompetitive inhibition, the slopes are independent of substrate concentration; with uncompetitive inhibition, the slopes of the lines decrease with increasing substrate concentrations. The kinetic parameters, Km, Ki, Ki', and beta (degree of partiality) can best be determined from respective secondary plots of slope and intercept vs substrate concentration, for competitive and noncompetitive inhibition mechanism or slope and intercept vs reciprocal substrate concentration for uncompetitive inhibition mechanism. Functional consequencs of these analyses are represented in terms of specific enzyme-inhibitor systems.     

Inhibition studies on the metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.

In an effort to identify a competitive inhibitor that can be used in future spectroscopic and crystallographic studies and to better understand the interaction of a mercaptoacetic acid-thiolester-containing compound with metallo-beta-lactamase L1 from Stenotrophomonas maltophilia, inhibition studies using two thiol-containing compounds were conducted. N-(2'-Mercaptoethyl)-2-phenylacetamide is a competitive inhibitor of L1 with a K(i) of 50 +/- 3 microM, and this compound is not a time-dependent inactivator of L1. N-Benzylacetyl-d-alanylthioacetic acid is a competitive inhibitor of L1 with a K(i) of 1.6 +/- 0.3 microM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric studies revealed that 2 mol of mercaptoacetate covalently bind to L1 upon incubation of the enzyme with N-benzylacetyl-d-alanylthioacetic acid; however, this covalently modified enzyme has the same activity as wild-type L1. Last, inhibition studies were used to demonstrate that 4-morpholinoethanesulfonic acid does not inhibit L1, even at concentrations up to 300 mM. This work identifies two possible competitive inhibitors which can be used in future structural studies and further demonstrates inhibitory heterogeneity among the metallo-beta-lactamases.     

A regulatory invertase from sugar cane leaf-sheaths

A soluble β-fructofuranosidase was isolated from sugar cane leaf-sheaths. The enzyme attacks sucrose with an activation energy of 5700 cal/mol above 30° and 17 000 cal/mol below 30°. The enzyme was inhibited by the reaction products. Glucose is a simple non-competitive inhibitor, but fructose is a competitive inhibitor. Kinetic studies using double reciprocal plots and replots of 1/K_i, slope vs inhibitor concentration showed that fructose binds to two interacting sites of the enzyme. Per cent residual activity plotted against inhibitor concentration, and Hill plots confirmed the regulatory properties of the invertase. n was found to be close to 2, the number of binding sites established with the double reciprocal method. The tissue and cellular levels of sucrose, fructose and glucose were measured. Fructose was found at inhibitory concentrations confirming that the activity of the enzyme is probably modulated by the hexose pool of the leaf-sheaths.     

The mechanism of rat liver fructose-1,6-bisphosphate inhibition by fructose-2,6-bisphosphate

It was found that a decrease in the activating cation (Mg2+) concentration below [A]0.5 causes the disappearance of cooperativity of the fructose 1.6-bisphosphatase substrate binding sites induced by high fructose 2.6-bisphosphate concentrations without any significant alteration in the extent of the enzyme inhibition. Under these conditions, a competitive type of inhibition (with respect to the substrate) is transformed into a non-competitive type with an increase in the fructose 2.6-bisphosphate concentration. The data obtained confirm the viewpoint that fructose 2.6-bisphosphate binds to the enzyme at two distinct sites, a catalytic and an allosteric ones, differing in their affinity for the inhibitor. It is supposed that the interaction between the allosteric fructose 2.6-bisphosphate binding site and the activator site occupied by Mg2+ is necessary for the cooperative response of the enzyme to the substrate.     

The interaction of fructose 2,6-bisphosphate with an allosteric site of rat liver fructose 1,6-bisphosphatase

Rat liver fructose 1,6-bisphosphatase can be protected against partial inactivation by N-ethylmaleimide by low concentrations of fructose 2,6-bisphosphate or high concentrations of fructose 1,6-bisphosphate. The partially inactivated enzyme has a much reduced sensitivity to high substrate inhibition and has lost the sigmoid component of the inhibition by fructose 2,6-bisphosphate; this compound is a simple linear competitive inhibitor of the modified enzyme. The results suggest that fructose 2,6-bisphosphate can bind to the enzyme at two distinct sites, the catalytic site and an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit by binding to the allosteric site.