Kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase

A new microcalorimetric method for recording the kinetic parameters k(cat), K(m) and K(i) of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic alpha-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic alpha-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic alpha-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site.     

Some kinetic studies on the mechanism of action of carnitine acetyltransferase

1. Michaelis constants for substrates of carnitine acetyltransferase have been shown to be independent of the concentration of second substrate present. This applies to the forward reaction between acetyl-l-carnitine and CoASH, and to the back reaction between l-carnitine and acetyl-CoA. 2. Product inhibition of both forward and back reactions has been studied. Evidence has been obtained for independent binding sites for l-carnitine and CoASH. Acetyl groups attached to either substrate occupy overlapping positions in space when the substrates are bound to the enzyme. 3. Possible reaction mechanisms involving the ordered addition of substrates have been excluded by determining kinetic constants in the presence and absence of added product. 4. d-Carnitine and acetyl-d-carnitine have been shown to inhibit competitively with respect to l-carnitine and acetyl-l-carnitine. 5. It is concluded that the mechanism of action of carnitine acetyltransferase involves four binary and two or more ternary enzyme complexes in rapid equilibrium with free substrates, the interconversion of the ternary complexes being the rate-limiting step. The possible intermediate formation of an acetyl-enzyme cannot be excluded, but this could only arise from a ternary complex.     

ERp57 is essential for efficient folding of glycoproteins sharing common structural domains

ERp57 is a member of the protein disulphide isomerase family of oxidoreductases, which are involved in native disulphide bond formation in the endoplasmic reticulum of mammalian cells. This enzyme has been shown to be associated with both calnexin and calreticulin and, therefore, has been proposed to be a glycoprotein-specific oxidoreductase. Here, we identify endogenous substrates for ERp57 by trapping mixed disulphide intermediates between enzyme and substrate. Our results demonstrate that the substrates for this enzyme are mostly heavily glycosylated, disulphide bonded proteins. In addition, we show that the substrate proteins share common structural domains, indicating that substrate specificity may involve specific structural features as well as the presence of an oligosaccharide side chain. We also show that the folding of two of the endogenous substrates for ERp57 is impaired in ERp57 knockout cells and that prevention of an interaction with calnexin or calreticulin perturbs the folding of some, but not all, substrates with multiple disulphide bonds. These results suggest a specific role for ERp57 in the isomerisation of non-native disulphide bonds in specific glycoprotein substrates.     

The effect of the presence of glucose on the fermentation of mannose by the anaerobic fungus Neocallimastix frontalis strain RE1

Abstract The disappearance of mannose and the formation of formate, acetate, lactate, ethanol and succinate by Neocallimastix frontalis strain RE1 occurred slowly when mannose was the only substrate present. When an equal quantity of glucose was present, the fermentation of mannose increased. Incubations with ¹³C-labelled mannose and glucose confirmed that the presence of both substrates resulted in increased product formation from mannose and reduced product formation from glucose. The relative proportions of products formed from the two substrates varied, possibly in part due to differences in the rates of growth of the fungus. The strains of N. frontalis able to utilize mannose may have a competitive advantage in the rumen ecosystem.     

Optical and chemical identification of kinetic steps in trypsin- and chymotrypsin-catalysed reactions

Previous interpretations of the mechanism of trypsin- and chymotrypsin-catalysed reactions in terms of two intermediates, the Michaelis complex and an acyl-enzyme, were based on steady-state studies and on the observation of individual steps under sub-optimum conditions. In the present paper new methods for the rapid analysis of chemical events and for the spectrophotometric detection of individual steps are applied to these two enzymes. These methods can be used to study reactions with specific amino acid ester substrates. It can be shown that there are at least three distinct steps between the Michaelis complex and the release of ethanol; the latter is likely to correspond to acyl-enzyme formation. The relative rates of these three steps are measured by rapid-flow techniques from observations of the displacement of chromophoric inhibitors and reactions with specific substrates containing chromophores, as well as from ethanol analyses during a single turnover of the enzyme reactions. It is concluded that the reactions of trypsin and chymotrypsin with their specific substrates involve the formation of a specially reactive conformation of the enzyme–substrate complex and that the rate constants involved in this rearrangement are at least as important for the overall reaction as those of the subsequent formation and decomposition of the acyl-enzyme.     

Modulatory effects on proteinase kinetics caused by association of both enzyme and substrate to heparin

Heparin is shown to produce modulatory effects on the amidolytic activity of trypsin, thrombin and plasmin with various synthetic peptide substrates. Simple Michaelis-Menten kinetics are observed in the absence of heparin. In its presence an enhancement effect is observed at low substrate concentrations, and an inhibitory effect is observed at high substrate concentrations. Other polyanions like dextran sulphate, phosvitin and inositol hexakisphosphate produces a similar effect. The modulatory effect of heparin is abolished when it binds cations. Co-binding of both substrate and enzyme to heparin seems to be a necessary requirement for the effect to occur. A model is proposed which can account semiquantitatively for the kinetics observed. It is suggested that the mechanism, which involves co-binding of substrate and enzyme in an competitive manner to a macromolecular structure, may be of primary importance as a regulatory mechanism in blood coagulation and fibrinolysis.     

Metabolism of rat brain mitochondria. Studies on the potassium ion-stimulated oxidation of pyruvate

1. Rat brain-cortex mitochondria were incubated in media containing 1, 5 or 100mm-K(+) in the presence of ADP, uncoupler (FCCP, carbonyl cyanide p-trifluoro-methoxyphenylhydrazone) or valinomycin while metabolizing pyruvate and malate, or acetylcarnitine and malate or glutamate and malate as substrates. Both the uptake of oxygen and disappearance of substrate were measured under these conditions. 2. With pyruvate and malate as substrate in the presence of both ADP and valinomycin, both the uptake of oxygen and disappearance of pyruvate increased markedly on increasing the K(+) content of the incubation medium from 5 to 100mm-K(+). However, in the presence of uncoupler (FCCP), although the oxygen uptake doubled little change was observed in the rate of disappearance of pyruvate on increasing the K(+) concentration. 3. Only small changes in uptake of substrate and oxygen were observed in the presence of ADP, uncoupler (FCCP) or valinomycin on increasing the K(+) concentration when acetylcarnitine+malate or glutamate+malate were used as substrates by brain mitochondria. 4. Further, increasing the K(+) concentration from 1 to 20mm when rat brain mitochondria were oxidizing a mixture of pyruvate and glutamate in the presence of malate and ADP caused a 30% increase in the respiration rate, 50% increase in the rate of disappearance of pyruvate and an 80% decrease in the rate of disappearance of glutamate. 5. Investigation of the redox state of the cytochromes and the nicotinamide nucleotides in various conditions with either pyruvate or acetylcarnitine as substrates suggested that the specific stimulation of metabolism of pyruvate by K(+) could not be explained by a general stimulation of the electron-transport system. 6. Low-amplitude high-energy swelling of rat brain mitochondria was investigated in both Na(+)- and K(+)-containing media. Swelling of brain mitochondria was much greater in the Na(+)-containing medium and in this medium, the addition of Mg(2+) caused a partial reversal of swelling together with an 85% decrease in the rate of utilization of pyruvate. However, in the K(+)-containing medium, the addition of Mg(2+), although also causing a reversal of swelling, did not affect the rate of disappearance of pyruvate. 7. Measurements of the ATP, NADH/NAD(+) and acetyl-CoA/CoA contents were made under various conditions and no evidence that K(+) concentrations affected these parameters was obtained. 8. The results are discussed in relationship to the physiological significance of the stimulation of pyruvate metabolism by K(+) in rat brain mitochondria. It is proposed that K(+) causes its effects by a direct stimulation of the pyruvate dehydrogenase complex.     

Hemolymph cholinesterase activity in the Mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (Dunker, 1853) that was exposed to adverse natural and anthropogenic conditions

The influence of habitat conditions on the activity, the structure of the substrate specificity (the ratio of the substrate hydrolysis rates), and the kinetic parameters of substrate hydrolysis due to the effect of hemolymph cholinesterase of the mussel Crenomytilus grayanus was studied. Mussels were collected from areas that are influenced by seasonal and stationary upwelling, as well as from a polluted area. Upwelling and anthropogenic pressure were shown to alter the structure of hemolymph cholinesterase substrate specificity in mussels, up to complete loss of the ability to catalyze the hydrolysis of propionyland butyrylthiocholine. It was established that during the seasonal upwelling the efficiency of the cholinergic process in mussels is provided by a wide range of effective concentrations of the substrates and by decreasing their affinity to the enzyme. Under the conditions of chronic anthropogenic pollution, the cholinesterase of the mussel hemolymph loses its ability to hydrolyze substrates other than acetylthiocholine.     

Conditions for the assay of glutamate semialdehyde aminotransferase that overcome the problem of substrate instability.

Investigations into the kinetic properties of glutamate semialdehyde aminotransferase, a key enzyme in the metabolic pathway leading to chlorophyll, are made difficult by the instability of the enzyme's substrate glutamate 1-semialdehyde. The rate of spontaneous disappearance of this compound from solution is shown to vary with the square of its concentration and to be pH-dependent. Thus using conditions appropriate to the assay of the enzyme, half of the substrate is lost from solution in a few minutes. Second-order rate constants for the reaction are determined and conditions are selected whereby the effects of the spontaneous reaction are rendered insignificant. The steady-state kinetic properties of the enzyme determined using these conditions are reported.     

Characteristics and Activity Changes of Proteolytic Enzymes in Apple Leaves during Autumnal Senescence

At least four different proteinases are present in senescing apple leaves (Malus domestica Borkh. cv. Golden Delicious) as determined by their pH optima, substrate specificity, and their reactivity to proteinase inhibitors. An enzyme active at pH 4.5 to 5.0 appears to be a sulfhydryl-dependent (iodoacetamide and phenylmercuric acetate-sensitive) endoproteinase, and degradation of the large subunit of ribulose bisphosphate carboxylase was observed only with this enzyme. It is tentatively concluded that this endoproteinase is responsible for the breakdown of ribulose bisphosphate carboxylase in vivo. However, the presence of more than one endoproteinase in apple leaves is suggested by the broad range of pH optima of the SH-dependent enzyme. Another enzyme active at pH 6.0 appears to be a carboxypeptidase, and was sensitive to phenylmethylsulfonylfluoride. This enzyme showed a strong hydrolytic activity against carbobenzoxyphenylalanylalanine. A sulfhydryl-dependent aminopeptidase and a second hydroxyl-dependent carboxypeptidase were active at pH 7.5

Total autolytic activity (the sulfhydryl-dependent endoproteinase) as measured by the disappearance of proteins decreased during the period of protein decline. Evidence is presented that the measured proteinase activity can be dependent on assay methods and substrates. While the disappearance of protein measures most of endo-type activity, the ninhydrin assay appears to measure exo-type activity preferentially.

     

Vitamin K-dependent carboxylase: influence of the "propeptide" region on enzyme activity

The liver microsomal vitamin K-dependent carboxylase catalyzes the post-translational conversion of specific glutamyl to gamma-carboxyglutamyl (Gla) residues in precursor forms of a limited number of proteins. These proteins contain an amino-terminal extension (propeptide) that is presumed to serve as an enzyme recognition site to assure their normal processing. The free, noncovalently bound propeptide has also been shown to stimulate the in vitro activity of this enzyme. This peptide has now been shown to lower the app Km of a low-molecular-weight Glu site substrate while having no influence on the app Km of the other substrates, vitamin KH2, O2, and CO2/HCO3-. Propeptide addition was shown to have no influence on the ratio of the two products of the enzyme, Gla and vitamin K-2,3-epoxide. Stimulation of carboxylase activity by the propeptide from human factor X was observed in a number of rat tissues and in the liver of a number of different species. Stability of the enzyme in crude microsomal preparations was greatly enhanced by the presence of propeptide. These observations are consistent with the hypothesis that this region of the protein substrates for the carboxylase not only serves an enzyme recognition or docking function but also modulates the activity of the enzyme by altering the affinity for one of its substrates.     

Co-operativity and enzymatic activity in polymer-activated enzymes. A one-dimensional piggy-back binding model and its application to the DNA-dependent ATPase of DNA gyrase

The binding of a ligand to a one-dimensional lattice in the presence of a second ("rider") ligand, which binds only to the first ligand (piggy-back binding), is studied. A model derived from this study is used to analyze the effects of co-operativity on the reaction rates of enzymes activated by polymeric cofactors that provide multiple binding sites for the enzyme. It is found that in the presence of strong co-operativity, the steady-state reaction rates of polymer-activated enzymes can be very different from the Michaelis-Menten paradigm. By adjusting the co-operativity parameters and the binding constants of the ligands, the model can generate apparent auto-catalytic enhancement by substrates at low substrate concentrations and apparent substrate inhibition at high substrate concentrations. The model is shown to be able to explain the differences in the rates of ATP hydrolysis by DNA gyrase in the presence of long versus short DNA molecules and in the presence of long DNA molecules at different gyrase to DNA ratios.     

Donor substrate regulation of transketolase.

The influence of substrates on the interaction of apotransketolase with thiamin diphosphate was investigated in the presence of magnesium ions. It was shown that the donor substrates, but not the acceptor substrates, enhance the affinity of the coenzyme either to only one active center of transketolase or to both active centers, but to different degrees in each, resulting in a negative cooperativity for coenzyme binding. In the absence of donor substrate, negative cooperativity is not observed. The donor substrate did not affect the interaction of the apoenzyme with the inactive coenzyme analogue, N3'-pyridyl-thiamin diphosphate. The influence of the donor substrate on the coenzyme-apotransketolase interaction was predicted as a result of formation of the transketolase reaction intermediate 2-(alpha,beta-dihydroxyethyl)-thiamin diphosphate, which exhibited a higher affinity to the enzyme than thiamin diphosphate. The enhancement of thiamin diphosphate's affinity to apotransketolase in the presence of donor substrate is probably one of the mechanisms underlying the substrate-affected transketolase regulation at low coenzyme concentrations.     

Mechanism of 3-methylaspartase probed using deuterium and solvent isotope effects and active-site directed reagents: identification of an essential cysteine residue.

The mechanism of the L-threo-3-methylaspartate ammonia-lyase (EC 4.3.1.2) reaction has been probed using deuterium and solvent isotope effects with three different substrates, (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-3-methylaspartic acid. Each substrate appears to form a covalent adduct with the enzyme through the amination of a dehydroalanine (DehydAla-173) residue. The true substrates are N-protonated and at low pH, the alkylammonium groups are deprotonated internally in a closed solvent-excluded pocket after K+ ion, an essential cofactor, has become bound to the enzyme. At high pH, the amino groups of the substrates are able to react with the dehydroalanine residue prior to K+ ion binding. This property of the system gives rise to complex kinetics at pH 9.0 or greater and causes the formation of dead-end complexes which lack Mg2+ ion, another essential cofactor. The enzyme-substrate adduct is subsequently deaminated in two elimination processes. Hydrazines act as alternative substrates in the reverse reaction direction in the presence of fumaric acid derivatives, but cause irreversible inhibition in their absence. Borohydride and cyanide are not inhibitors. N-Ethylmaleimide also irreversibly inactivates the enzyme and labels residue Cys-361. The inactivation process is enhanced in the presence of cofactor Mg2+ ions and Cys-361 appears to serve as a base for the removal of the C-3 proton from the natural substrate, (2S,3S)-3-methylaspartic acid. The dehydroalanine residue appears to be protected in the resting form of the enzyme by generation of an internal thioether cross-link. The binding of the substrate and K+ ion appear to cause a conformational change which requires hydroxide ion. This is linked to reversal of the thioether protection step and generation of the base for substrate deprotonation at C-3. The deamination reaction displays high reverse reaction commitments and independent evidence from primary deuterium isotope effect data indicates that a thiolate acts as the base for deprotonation at C-3.     

Lysosomal enzyme precursors in human fibroblasts. Activation of cathepsin D precursor in vitro and activity of beta-hexosaminidase A precursor towards ganglioside GM2

Precursors of cathepsin D and beta-hexosaminidase were isolated from secretions of human fibroblasts and their activity was studied with natural substrates. The immunoprecipitated precursor of cathepsin D, Mr 53000, was inactive with radioactive hemoglobin as substrate. At pH 3.8-4.2 an activation of the precursor took place, which was correlated by a reduction in size to Mr 51500. The observed cleavage of cathepsin D precursor in vitro resembles the autocatalytic activation of pepsinogen. The precursor of beta-hexosaminidase A is able to cleave the natural substrate GM2 ganglioside. This reaction, like that of the mature enzyme, depends on the presence of a protein activator, which interacts with the substrate and the enzyme.     

Characterization of the Magnitude and Mechanism of Aldehyde Oxidase-mediated Nitric Oxide Production from Nitrite

Aldehyde oxidase (AO) is a cytosolic enzyme with an important role in drug and xenobiotic metabolism. Although AO has structural similarity to bacterial nitrite reductases, it is unknown whether AO-catalyzed nitrite reduction can be an important source of NO. The mechanism, magnitude, and quantitative importance of AO-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The kinetics and magnitude of AO-dependent NO formation were characterized. In the presence of typical aldehyde substrates or NADH, AO reduced nitrite to NO. Kinetics of AO-catalyzed nitrite reduction followed Michaelis-Menten kinetics under anaerobic conditions. Under physiological conditions, nitrite levels are far below its measured K_m value in the presence of either the flavin site electron donor NADH or molybdenum site aldehyde electron donors. Under aerobic conditions with the FAD site-binding substrate, NADH, AO-mediated NO production was largely maintained, although with aldehyde substrates oxygen-dependent inhibition was seen. Oxygen tension, substrate, and pH levels were important regulators of AO-catalyzed NO generation. From kinetic data, it was determined that during ischemia hepatic, pulmonary, or myocardial AO and nitrite levels were sufficient to result in NO generation comparable to or exceeding maximal production by constitutive NO synthases. Thus, AO-catalyzed nitrite reduction can be an important source of NO generation, and its NO production will be further increased by therapeutic administration of nitrite.     

Substrate Specifity of Chymotrypsin. Study of Induced Strain by Molecular Mechanics

Acylenzyme intermediates, produced by transfer of the acyl portions of selected natural substrates onto the catalytic serine hydroxyl of the serine protease chymotrypsin, were modeled with the AMBER force field. The obtained structures were used to calculate interaction and deformation energies. A set of 32 geometry variables were extracted out of each structure. They describe deformation effects specific for each substrate. It is shown by statistical analyses, that the interaction and deformation energies correspond to measured substrate reactivities. The extracted geometry variables are able to reproduce this dependency through multivariante statistical methods. These analyses suggest that there exist specific deformations of both the substrate and the enzyme portion, which are related to substrate reactivity. The geometry changes observed for high specific substrates are interpreted in terms of mechanistical requirements of the enzymatic reaction. The obtained model validates the hypothesis of induced strain as possible source of substrate specifity of chymotrypsin.     

Separation of prolyl 3-hydroxylase and 4-hydroxylase activities and the 4-hydroxyproline requirement for synthesis of 3-hydroxyproline

Differences between prolyl 3-hydroxylase and prolyl 4-hydroxylase activities were found in their stimulation and inactivation by dithiothreitol and in their affinity to poly-L-proline linked to agarose. The two enzyme activities were separated by gel filtration, the results demonstrating that they are due to separate proteins. Comparison of [¹⁴C]proline-labelled protocollagen and the same protein when fully 4-hydroxylated as substrates indicated dependence of 3-hydroxyproline formation on the presence of 4-hydroxyproline. It is suggested that the main substrate sequence for 3-hydroxyproline synthesis is -Gly-Pro-4Hyp-Gly-.     

Enzymatic bromination of barbituric acid and some of its derivatives

Barbituric acid, 1-methyl- and 1,3-dimethylbarbituric acid, some of its 5-phenyl derivatives, and 5-chlorobarbituric acid are presented as new substrates for the bromoperoxidase isolated from the brown alga Ascophyllum nodosum. This enzyme is able to convert these substrates into the corresponding 5-bromo or 5,5-dibromo derivatives in good yields. Kinetic measurements show that the structure of the examined substrates has little or no effect on the enzymatic rate of bromination. However, at low substrate concentration the reaction rate depends on both the concentration of the organic substrate and the concentration of hydrogen peroxide. A mechanism is proposed for the reactions of bromoperoxidase with its substrates. These reactions involve the formation of free hypobromous acid which can either brominate the organic halogen acceptor or produce singlet oxygen by a competing reaction with hydrogen peroxide.     

Competition between hydrocarbon and barbiturate for spectral binding to hepatic cytochrome P-450. Inferences concerning spin state of the enzyme

The substrates hexobarbital and ethylbenzene have been shown to compete for the spectral binding site of phenobarbital-induced rat hepatic microsomal cytochrome p-450. The two substrates produce different delta Absmax values, and the presence of one substrate does not affect the delta Absmax of the other substrate and vice versa. The respective binding constants for the two substrates are similarly unaffected. The conclusion drawn from these observations is that, over the concentration ranges studied, there is no change in the availability of the enzyme as a result of substrate addition; the difference in delta Absmax apparently being due to varying abilities of different substrates to bring about a spin shift in the enzyme. Evidence is presented to indicate that differences between enzymes from untreated male rats and phenobarbital-treated male rats are attributable to differences in the enzyme itself and not to changes in the nature of the membrane brought about by phenobarbital administration, at least insofar as heat entropy compensation is concerned. The enthalpy-entropy compensation observed in the binding of a homologous series of barbiturates to the microsomal membrane as determined from the membrane concentration dependence of their binding constants is shown to agree surprisingly well with the direct determination performed by Sitar and Mannering.