Role of the flexible loop of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus in enzyme catalysis

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), a type I PRTase, from Tritrichomonas foetus, is a potential target for antitritrichomonal chemotherapy. Structural data on all the type I PRTases reveal a highly flexible, 11-14-amino acid loop, presumably covering the active site. With the exception of a highly conserved Ser-Tyr dipeptide, the other amino acids constituting the loop vary widely among different PRTases. The roles of the conserved Ser73 and Tyr74 residues in the loop and the dynamics of the loop in T. foetus HGXPRTase were investigated using site-directed mutants, stop-flow kinetics, chemical modification, and two-dimensional (1)H-(15)N heteronuclear NMR relaxation experiments. S73A, Y74F, and Y74E mutants of HGXPRTase exhibited a 5-7-fold increase in K(m) for guanine and a 3-5-fold increase in K(m) for PRPP compared to that of the wild type, reflecting the decreased affinity of binding for the two substrates. The k(cat)'s for these mutant-catalyzed reactions, however, do not change appreciably from that of the wild-type enzyme. Stopped-flow fluorescence with a Y74W mutant showed no apparent quenching by adding either PRPP or GMP alone. When both PRPP and guanine were added together, however, the fluorescence was rapidly quenched, followed by a slow recovery as the enzyme-catalyzed reaction progressed, suggesting movement of the loop during catalysis. In the presence of 9-deazaguanine and PRPP, the rapidly quenched fluorescence was not recovered, suggesting a closed loop form. The accessibility of Trp74 in the flexible loop of the mutant enzyme was also analyzed using N-bromosuccinimide (NBS), which reacts specifically with the tryptophan residue. NBS reacted with the only tryptophan in the Y74W mutant enzyme and rendered the enzyme inactive. GMP or PRPP alone failed to protect the enzyme from NBS inactivation. However, the presence of both 9-deazaguanine and PPRP protected the enzyme, allowing it to retain up to 70% of its activity. An S75H mutant, labeled with [(15)N]histidine, was used in the (1)H-(15)N NMR study. Spectra obtained in the presence of enzyme substrates indicated an apparent stabilization of the loop only in the presence of 9-deazaguanine and PRPP. These experimental results thus clearly demonstrated stabilization of the flexible loop upon binding of both PRPP and guanine and suggested its involvement in enzyme catalysis.     

Interdomain signaling in glutamine phosphoribosylpyrophosphate amidotransferase

The glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase-catalyzed synthesis of phosphoribosylamine from PRPP and glutamine is the sum of two half-reactions at separated catalytic sites in different domains. Binding of PRPP to a C-terminal phosphoribosyltransferase domain is required to activate the reaction at the N-terminal glutaminase domain. Interdomain signaling was monitored by intrinsic tryptophan fluorescence and by measurements of glutamine binding and glutamine site catalysis. Enzymes were engineered to contain a single tryptophan fluorescence reporter in key positions in the glutaminase domain. Trp(83) in the glutamine loop (residues 73-84) and Trp(482) in the C-terminal helix (residues 471-492) reported fluorescence changes in the glutaminase domain upon binding of PRPP and glutamine. The fluorescence changes were perturbed by Ile(335) and Tyr(74) mutations that disrupt interdomain signaling. Fluoresence titrations of PRPP and glutamine binding indicated that signaling defects increased the K(d) for glutamine but had little or no effect on PRPP binding. It was concluded that the contact between Ile(335) in the phosphoribosyltransferase domain and Tyr(74) in the glutamine site is a primary molecular interaction for interdomain signaling. Analysis of enzymes with mutations in the glutaminase domain C-terminal helix and a 404-420 peptide point to additional signaling interactions that activate the glutamine site when PRPP binds.     

Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase.

Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate. Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme. Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step. Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118). The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex. Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged.     

Quinolinate phosphoribosyltransferase: kinetic mechanism for a type II PRTase

Quinolinate phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) catalyzes the formation of nicotinate mononucleotide, carbon dioxide, and pyrophosphate from 5-phosphoribosyl 1-pyrophosphate (PRPP) and quinolinic acid (QA, pyridine 2,3-dicarboxylic acid). The enzyme is the only type II PRTase whose X-ray structure is known. Here we determined the kinetic mechanism of the enzyme from Salmonella typhimurium. Equilibrium binding studies show that PRPP and QA each form binary complexes with the enzyme, with K(D) values (53 and 21 microM, respectively) similar to their K(M) values (30 and 25 microM, respectively). Although neither PP(i) nor NAMN products bound well to the enzyme, 130-fold tighter binding of PP(i) (K(D) = 75 microM) and NAMN (K(D) = 6 microM) in a ternary complex was observed. Phthalic acid (K(D) = 21 microM) and PRPP each caused a 2.5-fold tightening of the other's binding. Isotope trapping experiments indicated that the E.QA complex is catalytically competent, whereas the E.PRPP complex could not be trapped. Pre-steady-state kinetics gave a linear rate of NAMN formation, indicating that on-enzyme phosphoribosyl transfer chemistry is rate-determining. Isotope trapping from the steady state revealed that nearly all QA and about one-third of PRPP in ternary enzyme.QA.PRPP complexes could be trapped as the product. Substrate inhibition by PRPP was observed. These data demonstrate a predominantly ordered kinetic mechanism in which productive binding of quinolinic acid precedes that of PRPP. An E.PRPP complex exists as a nonproductive side branch.     

Studies on pig muscle aldose reductase. Kinetic mechanism and evidence for a slow conformational change upon coenzyme binding.

Steady state kinetic analysis at pH 7.0 of the reduction of DL-glyceraldehyde by pig muscle aldose reductase showed that the enzyme follows a sequential ordered mechanism with NADPH binding first. However, the "off constant" for NADP+ in the forward direction was 1 order of magnitude less than the kcat. Analysis of this anomaly by pre-steady state kinetics using stopped-flow fluorescence spectroscopy showed that this could be accounted for by isomerization of the enzyme-NADP+ complex and that the rate of isomerization is the rate-limiting step. The rate constant for this step was of the same order of magnitude as the kcat for the forward reaction. Fluorescence emission spectra of free and NADP(H)-bound enzyme suggested a conformational change upon binding of coenzyme. In the reverse direction (oxidation of glycerol) pre-steady state and steady state kinetic analyses were consistent with the rate-limiting step occurring before isomerization of the enzyme-NADPH complex. We conclude, therefore, that during the kinetic mechanism of the reduction of aldehydes by aldose reductase, a slow (kinetically detectable) conformational change in the enzyme occurs upon coenzyme binding. Since NADPH and NADP+ bind to the enzyme very tightly, this has implications for the targeting and binding of drugs that are aldose reductase inhibitors.     

Catalytic cycle of the biosynthetic reaction catalyzed by adenylylated glutamine synthetase from Escherichia coli

The covalently attached AMP moiety of adenylylated glutamine synthetase from Escherichia coli has been replaced by its fluorescent analog, 2-aza-1,N6-etheno-AMP (aza-epsilon-AMP). The modified glutamine synthetase (aza-epsilon-GS) exhibits divalent cation requirement (Mn2+, rather than Mg2+), pH profile, Vmax, and Km similar to those of naturally adenylylated glutamine synthetase. Whereas naturally adenylylated glutamine synthetase exhibits only negligible fluorescence changes upon the binding of substrates, aza-epsilon-GS exhibits large fluorescence changes. The fluorescence changes have been used by means of a stopped flow technique to reveal the involvement of five fluorometrically distinct intermediates in the catalytic cycle for the biosynthesis of glutamine catalyzed by the adenylylated glutamine synthetase. The mechanism is very similar to that previously established for the unadenylylated enzyme, using intrinsic tryptophan fluorescence. Substrates bind via a rapid equilibrium random mechanism, but the reaction proceeds in a stepwise manner. The formation of an enzyme-bound intermediate (probably gamma-glutamyl phosphate + ADP) from ATP and L-glutamate is the rate-limiting step, with the subsequent reaction of the enzyme-bound intermediate occurring very rapidly. The success in elucidating this complex mechanism is due largely to the vastly different amplitudes of the fluorescence changes at the two excitation maxima (300 nm and 360 nm) of the aza-epsilon-AMP moiety which accompany the formation of the various intermediates.     

The adenine phosphoribosyltransferase from Giardia lamblia has a unique reaction mechanism and unusual substrate binding properties

Purine phosphoribosyltransferases catalyze the Mg2+ -dependent reaction that transforms a purine base into its corresponding nucleotide. They are present in a wide variety of organisms including plants, mammals, and parasitic protozoa. Giardia lamblia, the causative agent of giardiasis, lacks de novo purine biosynthesis and relies primarily on adenine and guanine phosphoribosyltransferases (APRTase and GPRTase) constituting two independent and essential purine salvage pathways. The APRTase from G. lamblia was cloned and expressed with a 6-His tag at its C terminus and purified to apparent homogeneity. Adenine and alpha-d-5-phosphoribosyl-1-pyrophosphate (PRPP) have K(m) values of 4.2 and 143 microm with a k(cat) of 2.8 s(-1) in the forward reaction, whereas AMP and PP(i) have K(m) values of 87 and 450 microm with a k(cat) of 9.5 x 10(-3) s(-1) in the reverse reaction. Product inhibition studies indicated that the forward reaction follows a random Bi Bi mechanism. Results from the kinetics of equilibrium isotope exchange further verified a random Bi Bi mechanism in the forward reaction. In a mutant enzyme, F25W, with kinetic constants similar to those of the wild type and a tryptophan residue at the adenine binding site, the addition of adenine or AMP to the free mutant enzyme resulted in fluorescence quenching, whereas PRPP caused fluorescence enhancement. The dissociation constants thus estimated are 16.5 microm for adenine, 14.3 microm for AMP, and 83.0 microm for PRPP. PP(i) exerted no detectable effect on the tryptophan fluorescence at all, suggesting a lack of PP(i) binding to the free enzyme. An ordered substrate binding in the reverse reaction with AMP bound first followed by PP(i) is thus postulated.     

Structural and Kinetic Studies of the Allosteric Transition in Sulfolobus solfataricus Uracil Phosphoribosyltransferase: Permanent Activation by Engineering of the C-Terminus

Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-α-1-diphosphate (PRPP) and uracil to uridine monophosphate (UMP) and diphosphate (PP_i). The tetrameric enzyme from Sulfolobus solfataricus has a unique type of allosteric regulation by cytidine triphosphate (CTP) and guanosine triphosphate (GTP). Here we report two structures of the activated state in complex with GTP. One structure (refined at 2.8-Å resolution) contains PRPP in all active sites, while the other structure (refined at 2.9-Å resolution) has PRPP in two sites and the hydrolysis products, ribose-5-phosphate and PP_i, in the other sites. Combined with three existing structures of uracil phosphoribosyltransferase in complex with UMP and the allosteric inhibitor cytidine triphosphate (CTP), these structures provide valuable insight into the mechanism of allosteric transition from inhibited to active enzyme. The regulatory triphosphates bind at a site in the center of the tetramer in a different manner and change the quaternary arrangement. Both effectors contact Pro94 at the beginning of a long β-strand in the dimer interface, which extends into a flexible loop over the active site. In the GTP-bound state, two flexible loop residues, Tyr123 and Lys125, bind the PP_i moiety of PRPP in the neighboring subunit and contribute to catalysis, while in the inhibited state, they contribute to the configuration of the active site for UMP rather than PRPP binding. The C-terminal Gly216 participates in a hydrogen-bond network in the dimer interface that stabilizes the inhibited, but not the activated, state. Tagging the C-terminus with additional amino acids generates an endogenously activated enzyme that binds GTP without effects on activity.     

Induced fit and kinetic mechanism of adenylation catalyzed by Escherichia coli threonyl-tRNA synthetase

Threonyl-tRNA synthetase (ThrRS) must discriminate among closely related amino acids to maintain the fidelity of protein synthesis. Here, a pre-steady state kinetic analysis of the ThRS-catalyzed adenylation reaction was carried out by monitoring changes in intrinsic tryptophan fluorescence. Stopped flow fluorimetry for the forward reaction gave a saturable fluorescence quench whose apparent rate increased hyperbolically with ATP concentration, consistent with a two-step mechanism in which rapid substrate binding precedes an isomerization step. From similar experiments, the equilibrium dissociation constants for dissociation of ATP from the E.Thr complex (K(3) = 450 +/- 180 microM) and threonine from the E.ATP complex (K'(4) = 135 microM) and the forward rate constant for adenylation (k(+5) = 29 +/- 4 s(-1)) were determined. A saturable fluorescence increase accompanied the pyrophosphorolysis of the E.Thr - AMP complex, affording the dissociation constant for PP(i) (K(6) = 170 +/- 50 microM) and the reverse rate constant (k(-5) = 47 +/- 4 s(-1)). The longer side chain of beta-hydroxynorvaline increased the apparent dissociation constant (K(4[HNV]) = 6.8 +/- 2.8 mM) with only a small reduction in the forward rate (k'(+5[HNV]) = 20 +/- 3.1 s(-1)). In contrast, two nonproductive substrates, threoninol and the adenylate analogue 5'-O-[N-(L-threonyl)sulfamoyl]adenosine (Thr-AMS), exhibited linear increases in k(app) with ligand concentration, suggesting that their binding is slow relative to isomerization. The proposed mechanism is consistent with steady state kinetic parameters. The role of threonine binding loop residue Trp434 in fluorescence changes was established by mutagenesis. The combined kinetic and molecular genetic analyses presented here support the principle of induced fit in the ThrRS-catalyzed adenylation reaction, in which substrate binding drives conformational changes that orient substrates and active site groups for catalysis.     

Dual role for the glutamine phosphoribosylpyrophosphate amidotransferase ammonia channel. Interdomain signaling and intermediate channeling

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalyzes the first reaction of de novo purine nucleotide synthesis in two steps at two sites. Glutamine is hydrolyzed to glutamate plus NH(3) at an N-terminal glutaminase site, and NH(3) is transferred through a 20-A hydrophobic channel to a distal PRPP site for synthesis of phosphoribosylamine. Binding of PRPP is required to activate the glutaminase site (termed interdomain signaling) to prevent the wasteful hydrolysis of glutamine in the absence of phosphoribosylamine synthesis. Mutations were constructed to analyze the function of the NH(3) channel. In the wild type enzyme, NH(3) derived from glutamine hydrolysis was transferred to the PRPP site, and little or none was released. Replacement of Leu-415 at the PRPP end of the channel with an alanine resulted in a leaky channel and release of NH(3) to the solvent. Mutations in five amino acids that line the channel and two other residues required for the reorganization of phosphoribosyltransferase domain "flexible loop" that leads to formation of the channel perturbed channel function as well as interdomain signaling. The data emphasize the role of the NH(3) channel in coupling interdomain signaling and NH(3) transfer.     

5-Phosphoribosylpyrophosphate amidotransferase from soybean root nodules: kinetic and regulatory properties

Most of the nitrogen transported from the nodules of nitrogen-fixing soybean plants is in the form of the ureides allantoin and allantoic acid. Recent work has shown that ureides are formed in the plant fraction of the nodule from de novo purine biosynthesis and purine oxidation. 5-Phosphoribosylpyrophosphate amidotransferase (PRAT), which catalyzes the first committed step of purine biosynthesis, has been purified 1500-fold from soybean root nodules. The enzyme had an apparent Mr of 8 X 10(6), but this estimate may have been for an aggregation of several purine biosynthetic activities. PRAT showed a pH optimum of pH 8.0, and Km values were 18 and 0.4 mM for glutamine and 5-phosphoribosyl-1-pyrophosphate (PRPP), respectively. The reaction required Mg2+, and PRPPMg3- was shown to be the reactive molecular species of PRPP. Ammonia could replace glutamine as a substrate, and the Vm with ammonia was twice that obtained when glutamine was the substrate. The initial-rate kinetics showed sequential addition of substrates to the enzyme. Product inhibition data was consistent with the order of product release being phosphoribosylamine, PPi, and glutamate. The enzyme was subject to regulation by end products of the purine biosynthetic pathway. IMP and GMP inhibited competitively with PRPP and promoted cooperativity in the binding of this substrate; there was no cooperativity in the binding of IMP to the enzyme. XMP was a linear competitive inhibitor with PRPP. The results are discussed in terms of the key regulatory point occupied by PRAT in the pathway of ureide biogenesis.     

The slow-onset nature of allosteric inhibition in α-isopropylmalate synthase from Mycobacterium tuberculosis is mediated by a flexible loop

The identification of structure-function relationships in allosteric enzymes is essential to describing a molecular mechanism for allosteric processes. The enzyme α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) is subject to slow-onset, allosteric inhibition by l-leucine. Here we report that alternate amino acids act as rapid equilibrium noncompetitive inhibitors of MtIPMS failing to display biphasic inhibition kinetics. Amino acid substitutions on a flexible loop covering the regulatory binding pocket generate enzyme variants that have significant affinity for l-leucine but lack biphasic inhibition kinetics. Taken together, these results are consistent with the flexible loop mediating the slow-onset step of allosteric inhibition.     

The crystal structure of TrpD, a metabolic enzyme essential for lung colonization by Mycobacterium tuberculosis, in complex with its substrate phosphoribosylpyrophosphate

Mycobacterium tuberculosis, the cause of tuberculosis, presents a major threat to human health worldwide. Biosynthetic enzymes that are essential for the survival of the bacterium, especially in activated macrophages, are important potential drug targets. Although the tryptophan biosynthesis pathway is thought to be non-essential for many pathogens, this appears not to be the case for M.tuberculosis, where a trpD gene knockout fails to cause disease in mice. We therefore chose the product of the trpD gene, anthranilate phosphoribosyltransferase, which catalyses the second step in tryptophan biosynthesis, for structural analysis. The structure of TrpD from M.tuberculosis was solved by X-ray crystallography, at 1.9 A resolution for the native enzyme (R = 0.191, Rfree = 0.230) and at 2.3 A resolution for the complex with its substrate phosphoribosylpyrophosphate (PRPP) and Mg2+ (R = 0.194, Rfree = 0.255). The enzyme is folded into two domains, separated by a hinge region. PRPP binds in the C-terminal domain, together with a pair of Mg ions. In the substrate complex, two flexible loops change conformation compared with the apo protein, to close over the PRPP and to complete an extensive network of hydrogen-bonded interactions. A nearby pocket, adjacent to the hinge region, is postulated by in silico docking as the binding site for anthranilate. A bound molecule of benzamidine, which was essential for crystallization and is also found in the hinge region, appears to reduce flexibility between the two domains.     

Subsite structure and ligand binding mechanism of glucoamylase

1. The basic concept and outline of the subsite theory were described, which correlates quantitatively the subsite structure (the arrangement of subsite affinities) to the action pattern of amylases in a unified manner. 2. The subsite structures of several amylases including glucoamylase were summarized. 3. In parallel with the theoretical prediction obtained therefrom, the binding subsites of glucose, gluconolactone and linear substrates to Rhizopus glucoamylase were investigated experimentally, by using steady-state inhibition kinetics, difference absorption spectrophotometry, and fluorometric titration. 4. From several lines of evidence, it was concluded that gluconolactone, a transition state analogue, is bound at Subsite 1 (nonreducing end side) where a tryptophan residue is located. 5. The stopped-flow kinetic studies have revealed that all the ligand bindings studied consist of two-step mechanism in which a bimolecular association between the enzyme and a ligand to form a loosely bound complex (EL) followed by the unimolecular isomerization process in which EL converts to the final firmly bound EL complex. For substrates the EL may be the productive complex and the fluorescence of the tryptophan located at Subsite 1 is quenched in their isomerization process, most probably a relocation of ligand to occupy this subsite.     

Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants.

Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding. Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule. Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent beta-lactamase and arylsulfatase. Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates. The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II. At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained. The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions. The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding. The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved "zinc binding motif" is located in a flexible region of the active site in which Zn(II) participates in catalysis.     

Stopped-flow studies of 2'-deoxynucleotide binding to thymidylate synthase

The mechanism of 2'-deoxynucleotide binding to Lactobacillus casei thymidylate synthase was studied using stopped-flow kinetic techniques to monitor the decrease in intrinsic protein fluorescence upon complex formation. The data were consistent with a two-step mechanism involving a rapid preequilibrium step to form the enzyme-2'-deoxynucleotide complex followed by a slow isomerization step. Rate and equilibrium constants were determined for the three 2'-deoxynucleotides (2'-deoxyuridylate, 2'-deoxythymidylate, and 5-fluoro-2'-deoxyuridylate) as a function of temperature. Similar free energy changes were found for all 2'-deoxynucleotides; however, the enthalpy and entropy changes for each step of the reaction differed for each 2'-deoxynucleotide. The thermodynamic profiles indicated that the isomerization step stabilized the enzyme-2'-deoxynucleotide complex by an additional 1500 cal/mol.     

Resolution of conformational states of Dictyostelium myosin II motor domain using tryptophan (W501) mutants: implications for the open-closed transition identified by crystallography

When myosin interacts with ATP there is a characteristic enhancement in tryptophan fluorescence which has been widely exploited in kinetic studies. Using Dictyostelium motor domain mutants, we show that W501, located at the end of the relay helix close to the converter region, responds to two independent conformational events on nucleotide binding. First, a rapid isomerization gives a small fluorescence quench and then a slower reversible step which controls the hydrolysis rate (and corresponds to the open-closed transition identified by crystallography) gives a large enhancement. A mutant lacking W501 shows no ATP-induced enhancement in the fluorescence, yet quenched-flow measurements demonstrate that ATP is rapidly hydrolyzed to give a products complex as in the wild-type. The nucleotide-free, open and closed states of a single tryptophan-containing construct, W501+, show distinct fluorescence spectra and susceptibilities to acrylamide quenching which indicate that W501 becomes internalized in the closed state. The open-closed transition does not require hydrolysis per se and can be induced by a nonhydrolyzable analogue. At 20 degrees C, the equilibrium may favor the open state, but with ATP as substrate, the subsequent hydrolysis step pulls the equilibrium toward the closed state such that a tryptophan mutant containing only W501 yields an overall 80% enhancement. These studies allow solution-based assays to be rationalized with the crystal structures of the myosin motor domain and show that three different states can be distinguished at the interface of the relay and converter regions.     

Influence of alpha-CH-->NH substitution in C8-CoA on the kinetics of association and dissociation of ligands with medium chain acyl-CoA dehydrogenase

We previously reported that the kinetic profiles for the association and dissociation of functionally diverse C(8)-CoA-ligands, viz., octanoyl-CoA (substrate), octenoyl-CoA (product), and octynoyl-CoA (inactivator) with medium chain acyl-CoA dehydrogenase (MCAD), were essentially identical, suggesting that the protein conformational changes played an essential role during ligand binding and/or catalysis [Peterson, K. L., Sergienko, E. E., Wu, Y., Kumar, N. R., Strauss, A. W., Oleson, A. E., Muhonen, W. W., Shabb, J. B., and Srivastava, D. K. (1995) Biochemisry 34, 14942-14953]. To ascertain the structural basis of the above similarity, we investigated the kinetics of association and dissociation of alpha-CH-->NH-substituted C(8)-CoA, namely, 2-azaoctanoyl-CoA, with the recombinant form of human liver MCAD. The rapid-scanning and single wavelength stopped-flow data for the binding of 2-azaoctanoyl-CoA to MCAD revealed that the overall interaction proceeds via two steps. The first (fast) step involves the formation of an enzyme-ligand collision complex (with a dissociation constant of K(c)), followed by a slow isomerization step (with forward and reverse rate constants of k(f) and k(r), respectively) with concomitant changes in the electronic structure of the enzyme-bound FAD. Since the latter step involves a concurrent change in the enzyme's tryptophan fluorescence, it is suggested that the isomerization step is coupled to the changes in the protein conformation. Although the overall binding affinity (K(d)) of the enzyme-2-azaoctanoyl-CoA complex is similar to that of the enzyme-octenoyl-CoA complex, their microscopic equilibria within the collision and isomerized complexes show an opposite relationship. These results coupled with the isothermal titration microcalorimetric studies lead to the suggestion that the electrostatic interaction within the enzyme site phase modulates the microscopic steps, as well as their corresponding ground and transition states, during the course of the enzyme-ligand interaction.