Inhibition of cytoplasmic aspartate aminotransferase from porcine heart by R and S isomers of aminooxysuccinate and hydrazinosuccinate

D- and L-aminooxysuccinate were synthesized and evaluated as inhibitors of cytoplasmic aspartate aminotransferase (EC 2.6.1.1) from porcine heart. L-Aminooxysuccinate was shown to be a slow binding inhibitor of the pyridoxal phosphate form of the enzyme with a Ki of 160 nM and a half-life of the inhibited complex of 8 min. Kinetic analysis revealed that inhibition followed a two-step mechanism in which the last step was rate-limiting. D-Aminooxysuccinate was not inhibitory up to a concentration of 0.1 mM. These compounds were compared to D- and L-hydrazinosuccinate, which are potent slow binding inhibitors of aspartate aminotransferase with Ki values of 1.5 and 0.5 nM, respectively. Models of all four analogs were built into the active site of the closed form of the enzyme. The energy-minimized conformations of both L-isomers bound to aspartate aminotransferase show better geometry for hydrogen bond and ion pair formation than do the corresponding D-isomers. The aldimine double bond formed by the L-isomers is not coplanar with the pyridoxal phosphate ring in accordance with the spectral properties of the inhibitor complexes that are characterized by broad absorbance bands. This lack of planarity was not evident for the models of D-hydrazinosuccinate and D-aminooxysuccinate.     

Slow- and tight-binding inhibition of aspartate aminotransferase by L-hydrazinosuccinate

The inhibition of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) by L-hydrazinosuccinate has been studied. The velocity of the enzyme reaction decreased with time when the reaction was initiated by the addition of enzyme to a mixture of the assay components and L-hydrazinosuccinate, while it increased slowly from a low level when a preincubated mixture of the enzyme and the inhibitor was added to the reaction mixture to initiate the reaction. Nearly 50% decrease in the initial reaction velocity was produced by a prolonged preincubation of the enzyme with the inhibitor, both at low concentrations of about 2 nM. These findings indicate that the inhibition is of the slow- and tight-binding type. The time-course of the reaction of the enzyme and the inhibitor, examined by the change in activity, was not in accord with single-step mechanisms, but rather appeared to follow biphasic kinetics. The inhibition could be fully reversed only in the presence of L-cysteine sulfinate or large excess of L-aspartate to convert the regenerated enzyme to its pyridoxamine form. The time-course of the reversal followed pseudo-first-order kinetics. Quantitative analysis of the experimental data has shown that the results are consistent with a mechanism of enzyme-inhibitor interaction which involves a reaction of two consecutive, reversible steps. The overall inhibition constant for L-hydrazinosuccinate was calculated to be approx. 0.2 nM.     

Binding of C5-dicarboxylic substrate to aspartate aminotransferase: implications for the conformational change at the transaldimination step

The mechanism for the reaction of aspartate aminotransferase with the C4 substrate, l-aspartate, has been well established. The binding of the C4 substrate induces conformational change in the enzyme from the open to the closed form, and the entire reaction proceeds in the closed form of the enzyme. On the contrary, little is known about the reaction with the C5 substrate, l-glutamate. In this study, we analyzed the pH-dependent binding of 2-methyl-l-glutamate to the enzyme and showed that the interaction between the amino group of 2-methyl-l-glutamate and the pyridoxal 5'-phosphate aldimine is weak compared to that between 2-methyl-l-aspartate and the aldimine. The structures of the Michaelis complexes of the enzyme with l-aspartate and l-glutamate were modeled on the basis of the maleate and glutarate complex structures of the enzyme. The result showed that l-glutamate binds to the open form of the enzyme in an extended conformation, and its alpha-amino group points in the opposite direction of the aldimine, while that of l-aspartate is close to the aldimine. These models explain the observations for 2-methyl-l-glutamate and 2-methyl-l-aspartate. The crystal structures of the complexes of aspartate aminotransferase with phosphopyridoxyl derivatives of l-glutamate, d-glutamate, and 2-methyl-l-glutamate were solved as the models for the external aldimine and ketimine complexes of l-glutamate. All the structures were in the closed form, and the two carboxylate groups and the arginine residues binding them are superimposable on the external aldimine complex with 2-methyl-l-aspartate. Taking these facts altogether, it was strongly suggested that the binding of l-glutamate to aspartate aminotransferase to form the Michaelis complex does not induce a conformational change in the enzyme, and that the conformational change to the closed form occurs during the transaldimination step. The hydrophobic residues of the entrance of the active site, including Tyr70, are considered to be important for promoting the transaldimination process and hence the recognition of the C5 substrate.     

The K258R mutant of aspartate aminotransferase stabilizes the quinonoid intermediate.

Lys-258 of aspartate aminotransferase forms a Schiff base with pyridoxal phosphate and is responsible for catalysis of the 1,3-prototropic shift central to the transamination reaction sequence. Substitution of arginine for Lys-258 stabilizes the otherwise elusive quinonoid intermediate, as assessed by the long wavelength absorption bands observed in the reactions of this mutant with several amino acid substrates. The external aldimine intermediate is not detectable during reactions of this mutant with amino acids, although the inhibitor alpha-methylaspartate does slowly and stably form this species. These results suggest that external aldimine formation is one of the rate-determining steps of the reaction. The pyridoxamine-5'-phosphate-like enzyme form (330-nm absorption maximum) is unreactive toward keto acid substrates, and the coenzyme bound to this species is not dissociable from the protein.     

High resolution crystal structures of human kynurenine aminotransferase‐I bound to PLP cofactor,and in complex with aminooxyacetate

In this study, we report two high‐resolution structures of the pyridoxal 5′ phosphate (PLP)‐dependent enzyme kynurenine aminotransferase‐I (KAT‐I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT‐I at 1.28 Å resolution, and the other with the general PLP‐dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP‐bound structures. We also report the inhibition of KAT‐1 by AOAA and aminooxy‐phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 μM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT‐I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.     

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 degrees C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K(i)* = 4.5 x 10(-7) mM. The respective inhibition constant of TCoBQ was equal to: K(i)* = 2.4 x 10(-6) mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.     

Strain and catalysis in aspartate aminotransferase

The notion of "ground-state destabilization" has been well documented in enzymology. It is the unfavourable interaction (strain) in the enzyme-substrate complex, and increases the k(cat) value without changing the k(cat)/K(m) value. During the course of the investigation on the reaction mechanism of aspartate aminotransferase (AAT), we found another type of strain that is crucial for catalysis: the strain of the distorted internal aldimine in the unliganded enzyme. This strain raises the energy level of the starting state (E+S), thereby reducing the energy gap between E+S and ES(++) and increasing the k(cat)/K(m) value. Further analysis on the reaction intermediates showed that the Michaelis complex of AAT with aspartate contains strain energy due to an unfavourable interaction between the main chain carbonyl oxygen and the Tyr225-aldimine hydrogen-bonding network. This belongs to the classical type of strain. In each case, the strain is reflected in the pK(a) value of the internal aldimine. In the historical explanation of the reaction mechanism of AAT, the shifts in the aldimine pK(a) have been considered to be the driving forces for the proton transfer during catalysis. However, the above findings indicate that the true driving forces are the strain energy inherent to the respective intermediates. We describe here how these strain energies are generated and are used for catalysis, and show that variations in the aldimine pK(a) during catalysis are no more than phenomenological results of adjusting the energy levels of the reaction intermediates for efficient catalysis.     

Porcine cytosolic aspartate aminotransferase reconstituted with [4'-13C]pyridoxal phosphate. pH- and ligand-induced changes of the coenzyme observed by 13C NMR spectroscopy

Apoenzyme samples of aspartate aminotransferase (AspAT) purified from the cytosolic fraction of pig heart were reconstituted with [4'-13C]pyridoxal 5'-phosphate (pyridoxal-P). The 13C NMR spectra of AspAT samples thus generated established the chemical shift of 165.3 ppm for C4' of the coenzyme bound as an internal aldimine with lysine 258 of the enzyme at pH 5. In the absence of ligands the chemical shift of C4' was shown to be pH dependent, shifting 5 ppm upfield to a constant value of 160.2 ppm above pH 8, the resulting pKa of 6.3 in agreement with spectrophotometric titrations. The addition of the competitive inhibitor succinate to the internal aldimine raises the pKa of the imine to 7.8, consistent with the theory of charge neutralization in the active site. In the presence of saturating concentrations of 2-methylaspartic acid the C4' signal of the coenzyme was shown to be invariant with pH and located at 162.7 ppm, midway between the observed chemical shifts of the protonated and unprotonated forms of the internal aldimine. The intermediate chemical shift of the external aldimine complex is thought to reflect the observation of an equilibrium mixture composed of roughly equal populations of the protonated ketoenamine and a dipolar anion species, corresponding to their respective spectral bands at 430 and 360-370 nm. Conversion to the pyridoxamine form was accomplished via reaction of the internal aldimine with L-cysteinesulfinate or by reduction with sodium borohydride, and the resulting C4' chemical shifts were identified by difference spectroscopy. Finally, the line widths of the C4' resonance under the various conditions were measured and qualitatively compared. The results are discussed in terms of the current mechanism and molecular models of the active site of AspAT.     

Kinetics of interaction of trypsin with an anionic inhibitor of trypsin BWI-1a from buckwheat seeds

The kinetics of binding of bovine trypsin to a proteinaceous inhibitor of trypsin from buckwheat seeds (BWI-1a) has been studied. The association rate constant (k(ass)) was 2.2 x 10(6) M-1 x sec-1 and the dissociation rate constant (k(off)) of the enzyme--inhibitor complex was 3.5 x 10(-3) sec-1; the inhibition constant Ki was 1.5 nM. The inhibitor BWI-1a is of the slow, tightly binding type. The mechanism of the inhibition of bovine trypsin by the trypsin inhibitor BWI-1a was studied. The mechanism of inhibition was found to involve two steps according to the kinetic data.     

Multi-step analysis of Hg2+ ion inhibition of jack bean urease

We performed a multi-step analysis of the inhibition of jack bean urease by Hg(2+) ions that included residual activity measurements after incubation of the enzyme with the metal ion, reactivation of Hg(2+)-inhibited urease, protection of urease with thiol reagents prior to incubation with Hg(2+), progress curve analysis, and spectroscopic assay of thiol groups in urease-Hg(2+) complexes with a cysteine selective agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Hg(2+) ions were found to form stable complexes with urease that could rapidly be reversed only by the treatment with dithiotreitol, and not by dilution or dialysis. The residual activity data interpreted in terms of the Hill equation revealed the multisite Hg(2+) inhibition of urease, and along with the DTNB thiol-assay they demonstrated the involvement in the reaction with Hg(2+) of six cysteine residues per enzyme subunit, including the active-site flap cysteine. The molar ratios of the inhibitor and enzyme imply that the inhibition consists of the formation of RSHgX complexes, X being a water molecule or an anion. The time-dependent Hg(2+) inhibitory action on urease determined in the system without enzyme preincubation was best described by slow-binding mechanism with the steady-state inhibition constant K(i) = 1.9 nM (+/-10%).     

Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial D-amino acid transaminase. Effects of exogenous amines on the slow formation of intermediates

In bacterial D-amino acid transaminase, Lys-145, which binds the coenzyme pyridoxal 5'-phosphate in Schiff base linkage, was changed to Gln-145 by site-directed mutagenesis (K145Q). The mutant enzyme had 0.015% the activity of the wild-type enzyme and was capable of forming a Schiff base with D-alanine; this external aldimine was formed over a period of minutes depending upon the D-alanine concentration. The transformation of the pyridoxal-5'-phosphate form of the enzyme to the pyridoxamine-5'-phosphate form (i.e. the half-reaction of transamination) occurred over a period of hours with this mutant enzyme. Thus, information on these two steps in the reaction and on the factors that influence them can readily be obtained with this mutant enzyme. In contrast, these reactions with the wild-type enzyme occur at much faster rates and are not easily studied separately. The mutant enzyme shows distinct preference for D- over L-alanine as substrates but it does so about 50-fold less effectively than the wild-type enzyme. Thus, Lys-145 probably acts in concert with the coenzyme and other functional side chain(s) to lead to efficient and stereochemically precise transamination in the wild-type enzyme. The addition of exogenous amines, ethanolamine or methyl amine, increased the rate of external aldimine formation with D-alanine and the mutant enzyme but the subsequent transformation to the pyridoxamine-5'-phosphate form of the enzyme was unaffected by exogenous amines. The wild-type enzyme displayed a large negative trough in the circular dichroic spectrum at 420 nm, which was practically absent in the mutant enzyme. However, addition of D-alanine to the mutant enzyme generated this negative Cotton effect (due to formation of the external aldimine with D-alanine). This circular dichroism band gradually collapsed in parallel with the transformation to the pyridoxamine-5'-phosphate enzyme. Further studies on this mutant enzyme, which displays the characteristics of the wild-type enzyme but at attenuated rates, may yield information on the factors controlling the stereochemistry of the reaction as well as on the catalytic steps of the transaminase pathway.     

Inhibition of 4-aminobutyrate transaminase by ethanolamine-O-sulfate]

The analysis of the interaction of ethanolamine-O-sulphate with 4-aminobutyrate transaminase revealed that the inhibitory effect is exerted upon the substrate subsite of the active site of the enzyme in aldimine form. The inhibition in irreversible. The inactivation rate versus pH-curve was shown to have a sigmoid character with inclination point at neutral pH. The study of inhibition kinetics by the Kitz and Wilson method revealed a complex inhibitory pattern compatible with a minimal two-step mechanism. Rate constant of inactivation was found to be equal to 0.22 min-1 and the value of the inhibitory constant--to 1.1-10(-2) M.     

An iso-random Bi Bi mechanism for adenylate kinase.

An iso-random Bi Bi mechanism has been proposed for adenylate kinase. In this mechanism, one of the enzyme forms can bind the substrates MgATP and AMP, whereas the other form can bind the products MgADP and ADP. In a catalytic cycle, the conformational changes of the free enzyme and the ternary complexes are the rate-limiting steps. The AP(5)A inhibition equations derived from this mechanism show theoretically that AP(5)A acts as a competitive inhibitor for the forward reaction and a mixed noncompetitive inhibitor for the backward reaction.     

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25°C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K_i* = 4.5 × 10^{? 7} mM. The respective inhibition constant of TCoBQ was equal to: K_i* = 2.4 × 10^{? 6} mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.     

Mechanism of the irreversible inhibition of aspartate aminotransferase by the bacterial toxin L-2-amino-4-methoxy-trans-3-butenoic acid.

The naturally occurring toxin L-2-amino-4-methoxy-trans-3-butenoic (AMB) acid irreversibly inhibits pyridoxal phosphate-linked aspartate aminotransferase. The inhibitor is a substrate for the enzyme, and as such is converted into a highly reactive intermediate which chemically reacts with an active site residue, thus irreversibly inactivating the enzyme. Enzymological and model studies on AMB are presented which enable one to determine the precise mechanism of action of this toxin. The mechanism involves Schiff base formation between the enzyme and toxin followed by alpha-C--H bond cleavage and aldimine isomerization to generate a bifunctional Michael acceptor. This molecule alkylates an active site residue by an addition and elimination route.     

The kinetic mechanism of acyl-CoA:lysolecithin acyltransferase from rabbit lung

Acyl-CoA:lysolecithin acyltransferase is a key enzyme in the deacylation-reacylation pathway of biosynthesis of molecular species of lecithin. However, the mechanism of the reaction has been little studied. In this paper, the kinetic mechanism of acyl-CoA:lysolecithin acyltransferase, partially purified from rabbit lung, is studied. The double-reciprocal plots of initial velocity vs substrate concentration gave two sets of parallel lines which fitted to a ping-pong equation with the following parameters: Km (palmitoyl-CoA) = 8.5 +/- 2 microM, Km (lysolecithin) = 61 +/- 16 microM, and V = 18 +/- 4 nmol/min/mg protein. Inhibition studies by substrates, alternate substrates, and products supported the ping-pong mechanism, although some nonclassical behavior was observed. Palmitoyl-CoA did not inhibit even at concentrations of 100 Km. In contrast, lysolecithin was a dead-end inhibitor with a dissociation constant of Ki = 930 +/- 40 microM. Alternate substrates and CoA showed alternate pathways for the reaction due to the formation of ternary complexes. Dipalmitoylphosphatidylcholine inhibition pointed to an isomerization of the free enzyme prior to the start of the reaction. From these results, an iso-ping-pong kinetic mechanism for lysolecithin acyltransferase is proposed. The kinetic steps of the reaction are correlated with previous chemical studies of the enzyme.     

Differential effects of temperature and hydrostatic pressure on the formation of quinonoid intermediates from L-Trp and L-Met by H463F mutant Escherichia coli tryptophan indole-lyase

Escherichia coli tryptophan indole-lyase (Trpase) is a bacterial pyridoxal 5'-phosphate (PLP)-dependent enzyme which catalyzes the reversible beta-elimination of l-Trp to give indole and ammonium pyruvate. H463F mutant E. coli Trpase (H463F Trpase) has very low activity with l-Trp, but it has near wild-type activity with other in vitro substrates, such as S-ethyl-l-cysteine and S-(o-nitrophenyl)-l-cysteine [Phillips, R. S., Johnson, N., and Kamath, A. V. (2002) Formation in vitro of Hybrid Dimers of H463F and Y74F Mutant Escherichia coli Tryptophan Indole-lyase Rescues Activity with l-Tryptophan, Biochemistry 41, 4012-4019]. The interaction of H463F Trpase with l-Trp and l-Met, a competitive inhibitor, has been investigated by rapid-scanning stopped-flow, high-pressure, and pressure jump spectrophotometry. Both l-Trp and l-Met bind to H463F Trpase to form equilibrating mixtures of external aldimine and quinonoid intermediates, absorbing at approximately 420 and approximately 505 nm, respectively. The apparent rate constant for quinonoid intermediate formation exhibits a hyperbolic dependence on l-Trp and l-Met concentration. The rate constant for quinonoid intermediate formation from l-Trp is approximately 10-fold lower for H463F Trpase than for wild-type Trpase, but the rate constant for reaction of l-Met is similar for H463F Trpase and wild-type Trpase. The temperature dependence of the rate constants for quinonoid intermediate formation reveals that both l-Trp and l-Met have similar values of DeltaH(++), but l-Met has a more negative value of DeltaS(++). Hydrostatic pressure perturbs the spectra of the H463F l-Trp and l-Met complexes, by shifting the position of the equilibria between different quinonoid and external aldimine complexes. Pressure-jump experiments show relaxations at 500 nm after rapid pressure changes of 100-400 bar with both l-Trp and l-Met. The apparent rate constants for relaxation of l-Trp, but not l-Met, show a significant increase with pressure. From these data, the value of DeltaV(++) for quinonoid intermediate formation from the external aldimine of l-Trp can be estimated to be -26.5 mL/mol, a larger than expected negative value for a proton transfer. These results suggest that there may be a contribution to the deprotonation reaction either from quantum mechanical tunneling or from a mechanical coupling of protein motion and proton transfer associated with the reaction of l-Trp, but not with l-Met.     

Involvement of heparin chain length in the heparin-catalyzed inhibition of thrombin by antithrombin III

The mechanism of the heparin-promoted reaction of thrombin with antithrombin III was investigated by using covalent complexes of antithrombin III with either high-affinity heparin (Mr = 15,000) or heparin fragments having an average of 16 and 12 monosaccharide units (Mr = 4,300 and 3,200). The complexes inhibit thrombin in the manner of active site-directed, irreversible inhibitors: (Formula: see text) That is, the inhibition rate of the enzyme is saturable with respect to concentration of complexes. The values determined for Ki = (k-1 + k2)/k1 are 7 nM, 100 nM, and 6 microM when the Mr of the heparin moieties are 15,000, 4,300, 3,200, respectively, whereas k2 (2 S-1) is independent of the heparin chain length. The bimolecular rate constant k2/Ki for intact heparin is 3 X 10(8) M-1 S-1 and the corresponding second order rate constant k1 is 6.7 X 10(8) M-1 S-1, a value greater than that expected for a diffusion-controlled bimolecular reaction. The bimolecular rate constants for the complexes with heparin of Mr = 4,300 and 3,200 are, respectively, 2 X 10(7) M-1 S-1 and 3 X 10(5) M-1 S-1. Active site-blocked thrombin is an antagonist of covalent antithrombin III-heparin complexes: the effect is monophasic and half-maximum at 4 nM of antagonist against the complex with intact heparin, whereas the effect is weaker against complexes with heparin fragments and not monophasic. We conclude that virtually all of the activity of high affinity, high molecular weight heparin depends on binding both thrombin and antithrombin III to heparin, and that the exceptionally high activity of heparin results in part from the capacity of thrombin bound nonspecifically to heparin to diffuse in the dimension of the heparin chain towards bound antithrombin III. Increasing the chain length of heparin results in an increased reaction rate because of a higher probability of interaction between thrombin and heparin in solution.     

Kinetic analysis of the inhibition of the epidermal growth factor receptor tyrosine kinase by Lavendustin-A and its analogue

Lavendustin-A was reported to be a potent tyrosine kinase inhibitor of the epidermal growth factor (EGF) receptor (Onoda, T., Iinuma, H., Sasaki, Y., Hamada, M., Isshibi, K., Naganawa, H., Takeuchi, T., Tatsuta, K., and Umezawa, K. (1989) J. Nat. Prod. 52, 1252-1257). Its inhibition kinetics was studied in detail using the baculovirus-expressed recombinant intracellular domain of the EGF receptor (EGFR-IC). Lavendustin-A (RG 14355) is a slow and tight binding inhibitor of the receptor tyrosine kinase. The pre-steady state kinetic analysis demonstrates that the inhibition corresponds to a two-step mechanism in which an initial enzyme-inhibitor complex (EI) is rapidly formed followed by a slow isomerization step to form a tight complex (EI*). The dissociation constant for the initial rapid forming complex is 370 nM, whereas the overall dissociation constant is estimated to be less than or equal to 1 nM. The difference between the two values is due to the tight binding nature of the inhibitor to the enzyme in EI*. The kinetic analysis using a preincubation protocol to pre-equilibrate the enzyme with the inhibitor in the presence of one substrate showed that Lavendustin-A is a hyperbolic mixed-type inhibitor with respect to both ATP and the peptide substrate, with a major effect on the binding affinities for both substrates. An analogue of Lavendustin-A (RG 14467) showed similar inhibition kinetics to that of Lavendustin-A. The results of the pre-steady state analysis are also consistent with the proposed two-step mechanism. The dissociation constant for the initial fast forming complex in this case is 3.4 microM, whereas the overall dissociation constant is estimated to be less than or equal to 30 nM. It is a partial (hyperbolic) competitive inhibitor with respect to ATP. Its inhibition is reduced to different extents by different peptide substrates, when the peptide is added to the enzyme simultaneously with the inhibitor. When studied with the least protective peptide, K1 (a peptide containing the major autophosphorylation site of the EGF receptor), RG 14467 acts as a hyperbolic noncompetitive inhibitor with respect to the peptide.     

Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase.

Investigations have been made of the slow, tight-binding inhibition by methotrexate of the reaction catalyzed by dihydrofolate reductase from Streptococcus faecium A. Quantitative analysis has shown that progress curve data are in accord with a mechanism that involves the rapid formation of an enzyme-NADPH-methotrexate complex that subsequently undergoes a relatively slow, reversible isomerization reaction. From the Ki value for the dissociation of methotrexate from the E-NADPH-methotrexate complex (23 nM) and values of 5.1 and 0.013 min-1 for the forward and reverse rate constants of the isomerization reaction, the overall inhibition constant for methotrexate was calculated to be 58 pM. The formation of an enzyme-methotrexate complex was demonstrated by means of fluorescence quenching, and a value of 0.36 muM was determined for its dissociation constant. The same technique was used to determine dissociation constants for the reaction of methotrexate with the E-NADP and E-NADPH complexes. The results indicate that in the presence of either NADPH or NADP there is enhancement of the binding of methotrexate to the enzyme. It is proposed that methotrexate behaves as a pseudosubstrate for dihydrofolate reductase.