The use of natural and unnatural amino acid substrates to define the substrate specificity differences of Escherichia coli aspartate and tyrosine aminotransferases.

The tyrosine (eTATase) and aspartate (eAATase) aminotransferases of Escherichia coli transaminate diacarboxylic amino acids with similar rate constants. However, eTATase exhibits approximately 10(2)-10(4)-fold higher second-order rate constants for the transamination of aromatic amino acids than does eAATase. A series of natural and unnatural amino acid substrates was used to quantitate specificity differences for these two highly related enzymes. A general trend toward lower transamination activity with increasing side-chain length (extending from aspartate to glutamate to alpha-aminoadipate) is observed for both enzymes. This result suggests that dicarboxylate ligands associate with the two highly related enzymes in a similar manner. The high reactivity of the enzymes with L-Asp and L-Glu can be attributed to an ion pair interaction between the side-chain carboxylate of the amino acid substrate and the guanidino group of the active site residue Arg 292 that is common to both enzymes. A strong linear correlation between side-chain hydrophobicity and transamination rate constants obtains for n-alkyl side-chain amino substrates with eTATase, but not for eAATase. The present kinetic data support a model in which eAATase contains one binding mode for all classes of substrate, whereas the active site of eTATase allows an additional mode that has increased affinity for hydrophobic amino acid.     

Kinetics of the reconstituted dicarboxylate carrier from rat liver mitochondria

The dicarboxylate carrier from rat liver mitochondria was purified by the Amberlite/hydroxyapatite procedure and reconstituted in egg yolk phospholipid vesicles by removing the detergent with Amberlite. The efficiency of reconstitution was optimized with respect to the ratio of detergent/phospholipid, the concentration of phospholipid and the number of Amberlite column passages. In the reconstituted system the incorporated dicarboxylate carrier catalyzed a first-order reaction of malate/phosphate exchange. V of the reconstituted malate/phosphate exchange was determined to be 6000 mumol/min per g protein at 25 degrees C. This value was independent of the type of substrate present at the external or internal space of the liposomes (malate, phosphate or malonate). The half-saturation constant was 0.49 mM for malate, 0.54 mM for malonate and 1.41 mM for phosphate. The activation energy of the exchange reaction was determined to be 95.8 kJ/mol. The transport was independent of the external pH in the range between pH 6 and 8.     

PMN elastases: a comparison of the specificity of human isozymes and the enzyme from other species toward substrates and inhibitors

The human elastases isolated from polymorphonuclear neutrophils (PMN) and purulent sputum displayed identical kinetic constants toward substrates and inhibitors. The elastases from the two sources yield identical N-terminal sequences and were recognized by antiserum prepared against human sputum elastase (HSE) isozyme-4 (I-4). The data support the proposal put forth by Twumasi and Liener (1977, J. Biol. Chem. 252, 1917-1926) that the human elastase from sputum is of PMN origin. PMN elastases from other species displayed kinetic constants toward both substrates and inhibitors significantly different from the human enzyme. Therefore, extrapolation of inhibitor profiles from these elastases to the human source should be avoided. Four groups of isozymes were resolved from HSE by FPLC. Only the most basic isozyme (I-4) was obtained as a single species. The isozymes displayed identical macroscopic kinetic constants toward several substrates and two classes of inhibitors. The similar partition ratios observed with a cephalosporin-derived inhibitor suggest that the microscopic rate constants are also identical. The data support the proposal suggested by Baugh and Travis (1976, Biochemistry 15, 836-841) that HLE isozymes differ only in carbohydrate content. Whatever the source of human PMN elastase heterogeneity, it does not result in heterogeneous catalytic properties. In addition, a new protein was identified in elastase preparations derived from human sputum. This protein displayed homology to serine proteases and properties suggesting that it is identical to azurocidin.     

The mitochondrial phosphate carrier reconstituted in liposomes is inhibited by doxorubicin

The phosphate carrier has been isolated from beef heart mitochondria in the presence of cardiolipin and reconstituted in asolectin vesicles. It has been found that 100 microM doxorubicin and 100 microM Br-daunomycin inhibit the unidirectional phosphate uptake in the reconstituted liposomes to the same extent as N-ethylmaleimide. The inhibition by Br-daunomycin is not due to covalent interaction with the carrier. The specific interaction between doxorubicin and cardiolipin is responsible for the inhibition of the phosphate carrier. Br-daunomycin interacts with 3 mitochondrial proteins of apparent Mr approximately 45 000, approximately 35 000 and approximately 30 000.     

Phosphate-induced efflux of adenine nucleotides from rat-heart mitochondria: evaluation of the roles of the phosphate/hydroxyl exchanger and the dicarboxylate carrier

Upon the addition of inorganic phosphate, isolated rat-heart mitochondria released endogenous adenine nucleotides. To elucidate the mechanism of this phosphate-induced efflux, we evaluated the relative roles of three inner mitochondrial membrane carriers: the adenine nucleotide translocase, the phosphate/hydroxyl exchanger, and the dicarboxylate carrier. Atractyloside (a specific inhibitor of the adenine nucleotide translocase) prevented this efflux, but did not inhibit mitochondrial swelling. Inhibitors of the phosphate/hydroxyl exchanger (200 microM n-ethylmaleimide and 10 microM mersalyl) did not inhibit phosphate-induced efflux. 200 microM mersalyl (which inhibited both the phosphate/hydroxyl exchanger and the dicarboxylate carrier) inhibited the rate of efflux approx. 65% Phenylsuccinate and 2-n-butylmalonate (inhibitors of the dicarboxylate carrier) partially inhibited phosphate-induced efflux and adenine nucleotide translocase activity. Mersalyl (200 microM) had no effect on adenine nucleotide translocase activity. Partial inhibition of the adenine nucleotide translocase by phenylsuccinate and butylmalonate could not explain the extent of inhibition of phosphate-efflux by these agents. Moreover, the rates of adenine nucleotide efflux in the presence of phenylsuccinate, butylmalonate, or mersalyl correlated well with the ability of these agents to inhibit succinate-supported respiration. We conclude that phosphate-induced efflux of adenine nucleotides from rat heart mitochondria occurs over the adenine nucleotide translocase, and that the site of action of the phosphate is not the phosphate/hydroxyl exchanger, but is likely the dicarboxylate carrier.     

On porcine pancreatic alpha-amylase action: kinetic evidence for the binding of two maltooligosaccharide molecules (maltose, maltotriose and o-nitrophenylmaltoside) by inhibition studies. Correlation with the five-subsite energy profile

Hydrolysis of small substrates (maltose, maltotriose and o-nitrophenylmaltoside) catalysed by porcine pancreatic alpha-amylase was studied from a kinetic viewpoint over a wide range of substrate concentrations. Non-linear double-reciprocal plots are obtained at high maltose, maltotriose and o-nitrophenylmaltoside concentrations indicating typical substrate inhibition. These results are consistent with the successive binding of two molecules of substrate per enzyme molecule with dissociation constants Ks1 and Ks2. The Hill plot, log [v/(V-v)] versus log [S], is clearly biphasic and allows the dissociation constants of the ES1 and ES2 complexes to be calculated. Maltose and maltotriose are inhibitors of the amylase-catalysed amylose and o-nitrophenylmaltoside hydrolysis. The inhibition is of the competitive type. The (apparent) inhibition constant Kiapp varies with the inhibitor concentration. These results are also consistent with the successive binding of at least two molecules of maltose or maltotriose per amylase molecule with the dissociation constants Ki1 and Ki2. These inhibition studies show that small substrates and large polymeric ones are hydrolysed at the same catalytic site(s). The values of the dissociation constants Ks1 and Ki1 of the maltose-amylase complexes are identical. According to the five-subsite energy profile previously determined, at low concentration, maltose (as substrate and as inhibitor) binds to the same two sites (4,5) or (3,4), maltotriose (as substrate and as inhibitor) and o-nitrophenyl-maltoside (as substrate) bind to the same three subsites (3,4,5). The dissociation constants Ks2 and Ki2 determined at high substrate and inhibitor concentration are consistent with the binding of the second ligand molecule at a single subsite. The binding mode of the second molecule of maltose (substrate) and o-nitrophenylmaltoside remains uncertain, very likely because of the inaccuracy due to simplifications in the calculations of the subsite binding energies. No binding site(s) outside the catalytic one has been taken into account in this model.     

Purification and reconstitution of two anion carriers from rat liver mitochondria: the dicarboxylate and the 2-oxoglutarate carrier

Two anion-transporting systems, i.e., the dicarboxylate carrier and the 2-oxoglutarate carrier, have been purified from rat liver mitochondria and functionally identified. The dicarboxylate carrier has been isolated in active form by hydroxyapatite chromatography after partial removal of the solubilizing detergent Triton X-114 from the mitochondrial extract. The SDS gel electrophoresis of this preparation consists mainly of one protein band with an apparent Mr of 28,000, identified as the dicarboxylate carrier. Complete purification of the 28 kDa protein in inactive form has been achieved by sequential chromatography on hydroxyapatite and Celite followed by SDS extraction of the retained protein. The 2-oxoglutarate carrier has been purified by hydroxyapatite chromatography after extensive removal of Triton X-114 from the detergent extract. SDS gel electrophoresis of the purified fraction shows a single band with an apparent Mr of 32,500. When reconstituted into liposomes, the functional properties of the two isolated carrier proteins resemble closely those of the dicarboxylate and the 2-oxoglutarate transport systems characterized in mitochondria.     

Functional properties of purified and reconstituted mitochondrial metabolite carriers

Eight mitochondrial carrier proteins were solubilized and purified in the authors' laboratories using variations of a general procedure based on hydroxyapatite and Celite chromatography. The molecular mass of all the carriers ranges between 28 and 34 kDa on SDS-PAGE. The purified carrier proteins were reconstituted into liposomes mainly by using a method of detergent removal by hydrophobic chromatography on polystyrene beads. The various carriers were identified in the reconstituted state by their kinetic properties. A complete set of basic kinetic data including substrate specificity, affinity, interaction with inhibitors, and activation energy was obtained. These data closely resemble those of intact mitochondria, as far as they are available from the intact organelle. Mainly on the basis of kinetic data, the asymmetric orientation of most of the reconstituted carrier proteins were established. Several of their functional properties are significantly affected by the type of phospholipids used for reconstitution. All carriers which have been investigated in proteoliposomes function according to a simultaneous (sequential) mechanism of transport; i.e., a ternary complex, made up of two substrates and the carrier protein, is involved in the catalytic cycle. The only exception was the carnitine carrier, where a ping-pong mechanism of transport was found. By reaction of particular cysteine residues with mercurial reagents, several carriers could be reversibly converted to a functional state different from the various physiological transport modes. This unphysiological transport mode is characterized by a combination of channel-type and carrier-type properties.     

Reactivity of the -SH groups of the mitochondrial phosphate carrier under native, solubilized and reconstituted conditions

The isolated and liposome-reconstituted mitochondrial phosphate carrier exhibits a sigmoidal inhibition curve by mersalyl, similar to that found with intact mitochondria. In contrast a hyperbolic inhibition curve is found (a) by titration of the soluble carrier with mersalyl before reconstitution in liposomes and (b) by titration of the reconstituted carrier with mersalyl after successively pretreatment of the mitochondria with low, non-inhibitory concentrations of mersalyl, excess N-ethylmaleimide and dithiothreitol. The inhibition of the reconstituted, but not of the soluble, phosphate carrier by mersalyl can be reversed by dithiothreitol. Cupric di(1,10-phenanthroline) inhibits the soluble but not the reconstituted phosphate carrier. The inhibited phosphate carrier can be reactivated by dithiothreitol in the soluble state but not after reconstitution in liposomes. The data support the previously suggested model of the phosphate carrier, assuming a dimer of two identical subunits for the active unit.     

The relationship between substrate dissociation constants derived from transport experiments and from equilibrium binding assays. Implications of the conventional carrier model

A kinetic analysis of substrate and inhibitor binding, based on the conventional carrier model, leads to the following conclusions. The substrate constant derived from equilibrium binding studies is not a simple dissociation constant; rather, it is identical to the half-saturating substrate concentration for equilibrium exchange transport, which is a function of both the dissociation constant and the rate constants for carrier reorientation. In general, binding and transport constants are identical, assuming the same substrate distribution across the membrane in the two experiments. Binding studies reveal only a single substrate site--even if the carrier is unsymmetrical, with different substrate affinities on the two sides of the membrane. The binding constants for inhibitors are identical to the inhibition constants found in transport. These rules, which apply to a carrier imbedded in the cell membrane or free in solution, offer a means of deciding whether an isolated carrier retains the properties of the intact system.     

Enzymatic synthesis of the 2',5'-A4 tetramer analog, 2',5'-ppp3'dA(p3'dA)3, by rabbit reticulocyte lysates: binding and activation of the 2',5'-A eta dependent nuclease, hydrolysis of mRNA, and inhibition of protein synthesis

The exchange between external [¹⁴C] malonate and internal malate or phosphate was reconstituted in liposomes prepared by incorporation of a Triton-extract of mitochondrial rat-liver inner membranes. The conditions of transport were investigated and the kinetic parameters of malonate-malate and malonate-phosphate exchanges were determined. The exchange was sensitive to butylmalonate and to organomercurials. Sulfate and phosphate decreased the rate of malonatemalate exchange and phosphate inhibition was found to be competitive. This report demonstrates the possibility to reconstitute a functional dicarboxylate transport and to use the system for further purification and for studies at the molecular level.     

Asymmetric orientation of the reconstituted aspartate/glutamate carrier from mitochondria

A partially purified preparation of the aspartate/glutamate carrier from bovine heart mitochondria was reconstituted into liposomal membranes by chromatography on hydrophobic ion exchange resins. Based on the favorable conditions of this reconstituted system the transmembrane orientation of the inserted carrier protein could be determined by functional analysis. For reliable measurement of the reconstituted aspartate-glutamate exchange activity an optimized inhibitor-stop technique using pyridoxal phosphate was developed. By simultaneous application of both forward and backward exchange experiments the practical usefulness of the reconstituted system could be extended to investigations including variation of internal and external substrate concentrations over a wide range. Thereby a complete set of Km values for both aspartate and glutamate at both the internal and external side of the proteoliposomes could be established. These experiments led to the following results and conclusions: (i) The observed substrate affinities are clearly different for the two different membrane sides both for aspartate (external 50 microM, internal 3 mM) and glutamate (external about 200 microM, internal 3 mM). (ii) The exclusive presence of only one type of transport affinity for every single substrate at one side of the liposomal membrane clearly demonstrates the asymmetric orientation of the functionally active carrier protein molecules. (iii) When comparing the values of these constants with published data obtained in mitochondria, an inside-out orientation of the aspartate/glutamate carrier after isolation and reinsertion into liposomes is strongly suggested.     

Substrate inhibition kinetic model for West Nile virus NS2B-NS3 protease

West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested that peptide substrates could bind to two binding sites on the protease. Reaction product analogues also showed inhibition of the protease, demonstrating product inhibition in addition to and distinct from substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1'-P3' interaction surface (i.e., the S1'-S3' sites). Optimization of substrate analogue inhibitors that target these two independent sites may lead to novel anti-WNV drugs.     

P-Glycoprotein (P-gp) expressed in a confluent monolayer of hMDR1-MDCKII cells has more than one efflux pathway with cooperative binding sites

The multidrug resistance transporter P-glycoprotein (P-gp) effluxes a wide range of substrates and can be affected by a wide range of inhibitors or modulators. Many studies have presented classifications for these binding interactions, within either the context of equilibrium binding or the Michaelis-Menten enzyme analysis of the ATPase activity of P-gp. Our approach is to study P-gp transport and its inhibition using a physiologically relevant confluent monolayer of hMDR1-MDCKII cells. We measure the elementary rate constants for P-gp efflux of substrates and study inhibition using pairwise combinations with a different unlabeled substrate acting as the inhibitor. Our current kinetic model for P-gp has only a single binding site, because a previous study proved that the mass-action kinetics of efflux of a single substrate were not sensitive to whether there are one or more substrate-binding and efflux sites. In this study, using this one-site model, we found that, with "high" concentrations of either a substrate or an inhibitor, the elementary rate constants fitted independently for each of the substrates alone quantitatively predicted the efflux curves, simply applying the assumption that binding at the "one site" was competitive. On the other hand, at "low" concentrations of both the substrate and inhibitor, we found no inhibition of the substrate efflux, despite the fact that both the substrate and inhibitor were being well-effluxed. This was not an effect of excess "empty" P-gp molecules, because the competitive efflux model takes site occupancy into account. Rather, it is quantitative evidence that the substrate and inhibitor are being effluxed by multiple pathways within P-gp. Remarkably, increasing the substrate concentration above the "low" concentration, caused the inhibition to become competitive; i.e., the inhibitor became effective. These data and their analysis show that the binding of these substrates must be cooperative, either positive or negative.     

Characteristics of the interaction between 2-alkylmalonates and the substrate-binding site of the dicarboxylate carrier of rat liver mitochondria

The effect of thirteen synthetic 2-n-alkyl derivatives of malonic acid on the rate of the mitochondrial succinate oxidase reaction controlled by the dicarboxylate carrier, was studied. These compounds were shown to act as competitive inhibitors of succinate transport and could interact with the carrier according to the 1:1 stoichiometry. The affinity of the inhibitor for the carrier increased in parallel with the increase in the alkyl residue length. This dependence was impaired only in the case of pentyl-, hexyl- and heptylmalonates having close values of inhibition constants. The data obtained suggest that the polar site and two hydrophobic regions are located in the vicinity of the carrier active site. The possible organization of the carrier substrate-binding site is discussed.     

In vivo significance of kinetic constants of protein proteinase inhibitors

We describe the in vivo significance of the kinetic parameters which characterize the interaction between proteinases and protein proteinase inhibitors. Knowledge of the second-order association rate constant kass and in vivo inhibitor concentration allows the calculation of the delay time of inhibition, i.e., the time required for complete inhibition of a proteinase in vivo. The influence of biological substrates on the delay time is also analyzed. The extent of substrate breakdown during the delay time of inhibition may be computed from the various constants describing the proteinase/substrate/inhibitor interactions and the biological concentrations of proteinase and inhibitor. The in vivo partition of a proteinase between two inhibitors may be calculated if the kinetic parameters are known. We define a stability time for enzyme-inhibitor complexes as a minimal time during which the complexes may be considered as stable. This time is related to kdiss the dissociation rate constant of the reversible enzyme-inhibitor complex or to k, the breakdown rate constant of the complex formed with temporary inhibitors. The overall stability of the complex depends upon the ratio between the inhibitor concentration and Ki, the equilibrium dissociation constant of the complex. If this ratio is higher than 1000, a reversible inhibitor behaves like an irreversible one in vivo whatever the enzyme concentration.     

Kinetic mechanism of antiports catalyzed by reconstituted ornithine/citrulline carrier from rat liver mitochondria

The transport mechanism of the reconstituted ornithine/citrulline carrier purified from rat liver mitochondria was investigated kinetically. A complete set of half-saturation constants (K(m)) was established for ornithine, citrulline and H(+) on both the external and internal side of the liposomal membrane. The internal affinity for ornithine was much lower than that determined on the external surface. The exclusive presence of a single transport affinity for ornithine on each side of the membrane indicated a unidirectional insertion of the ornithine/citrulline carrier into liposomes, probably right-side-out with respect to mitochondria. Two-reactant initial velocity studies of the homologous (ornithine/ornithine) and heterologous (ornithine/citrulline) exchange reactions resulted in a kinetic pattern which is characteristic of a simultaneous antiport mechanism. This type of mechanism implies that the carrier forms a ternary complex with the substrates before the transport reaction occurs. A quantitative analysis of substrate interaction revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrates.     

Characterization of mannosyl-transfer reactions catalyzed by dolichyl-mannosyl-phosphate-synthase

Evidence suggesting that a single enzyme catalyzes mannosyl transfer from GDP-mannose to both dolichyl phosphate and to phenyl phosphate was obtained as follows: (a) The two activities were coeluted from columns of DEAE-cellulose and Sepharose CL-6B, (b) both reactions demonstrated similar kinetic constants for the glycosyl donor and for guanosine nucleoside inhibitors, (c) both reactions were sensitive to inhibition by low concentrations of nonionic detergents, and (d) both activities were found to be thermally inactivated at similar rates upon incubation at 55 degrees. The reaction conditions required for optimal mannosyl transfer by the purified enzyme preparation to the hydrophobic and water soluble acceptors, however, were found to be quite different. Whereas mannosyl transfer from GDP-mannose to dolichyl phosphate occurred at maximal rates only in the presence of specific phospholipids, the rate of mannosyl transfer to phenyl phosphate was essentially unaffected by the addition of phospholipid. These results indicate that dolichyl-mannosyl-phosphate-synthase, which has some of the properties of an intrinsic membrane protein, does not have an absolute requirement for phospholipid for catalytic activity, but rather that phospholipid is required for interaction of the enzyme with the long chain polyisoprenol substrate dolichyl phosphate.     

The competition plot: A kinetic method to assess whether an enzyme that catalyzes multiple reactions does so at a unique site

Enzymes often act on more than one substrate, and the question then arises as to whether this can be attributed to the existence of two different enzymes that have not been separated or, more interesting, to the presence of two different active sites in the same enzyme. The competition plot is a kinetic method that allows us to test with little experimentation whether the two reactions occur at the same site or at different sites. It consists of making mixtures of the two substrates and plotting the total rate against a parameter p that defines the concentrations of the two substrates in terms of reference concentrations chosen to give the same rates at p = 0 and p = 1, i.e., when only one of the substrates is present. With a slight modification of the equations it can also be applied to enzymes that deviate from Michaelis-Menten kinetics. If the two substrates react at the same site, the competition plot gives a horizontal straight line; i.e., the total rate is independent of p. In contrast, if the two reactions occur at two separate and independent sites a curve with a maximum is obtained; separate reactions with cross-inhibition generate curves with either maxima or minima according to whether the Michaelis constants of the two substrates are smaller or larger than their inhibition constants in the other reactions. Strategies to avoid ambiguous results and to improve the sensitivity of the plot are described. A practical example is given to facilitate the experimental protocol for this plot.     

Multiple sites and synergism in the binding of inhibitors to microsomal aminopeptidase

The active site of microsomal aminopeptidase has been probed by studying the inhibition of the enzyme in the simultaneous presence of two ligands. The results have been analyzed with the Yonetani-Theorell plot to quantitate the degree of interaction between the two inhibitors. As expected, the enzyme contains a strong binding site for the alpha-amino group and the hydrophobic side chain of specific substrates. In addition, however, the enzyme can interact with another amine and a second hydrophobic group. Evidence suggests that this extra amine may bind to the zinc in an unprotonated form and that one of the hydrophobic sites is located in the vicinity. Another unexpected finding in this work is a strong synergism between the binding of ammonia and that of zinc ligands such as hydroxamates. This synergism may reflect an induced-fit mechanism that brings the catalytically important zinc atom into the optimal state only in the presence of specific substrates.