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E W Miles

Publications and source records attributed to E W Miles.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cryo-crystallography of a true substrate, indole-3-glycerol phosphate, bound to a mutant (alphaD60N) tryptophan synthase alpha2beta2 complex reveals the correct orientation of active site alphaGlu49.

The reversible cleavage of indole-3-glycerol by the alpha-subunit of tryptophan synthase has been proposed to be catalyzed by alphaGlu49 and alphaAsp60. Although previous x-ray crystallographic structures of the tryptophan synthase alpha2beta2 complex showed an interaction between the carboxylate of alphaAsp60 and the bound inhibitor indole-3-propanol phosphate, the carboxylate of alphaGlu49 was too distant to play its proposed role. To clarify the structural and functional roles of alphaGlu49, we have determined crystal structures of a mutant (alphaD60N) alpha2beta2 complex in the presence and absence of the true substrate, indole-3-glycerol phosphate. The enzyme in the crystal cleaves indole-3-glycerol phosphate very slowly at room temperature but not under cryo-conditions of 95 K. The structure of the complex with the true substrate obtained by cryo-crystallography reveals that indole-3-glycerol phosphate and indole-3-propanol phosphate have similar binding modes but different torsion angles. Most importantly, the side chain of alphaGlu49 interacts with 3-hydroxyl group of indole-3-glycerol phosphate as proposed. The movement of the side chain of alphaGlu49 into an extended conformation upon binding the true substrate provides evidence for an induced fit mechanism. Our results demonstrate how cryo-crystallography and mutagenesis can provide insight into enzyme mechanism.

Amino Acid Sequence↗
PubMed1998

Mutations in the contact region between the alpha and beta subunits of tryptophan synthase alter subunit interaction and intersubunit communication.

Interaction between the alpha and beta subunits of tryptophan synthase leads to mutual stabilization of the active conformations and to coordinated control of the activities of the two subunits. To elucidate the roles of specific residues in the interaction site between the alpha and beta subunits, mutant alpha and beta subunits were constructed, and the effects of mutation on subunit interaction and intersubunit communication were determined. Mutation of either alpha subunit Asp56 (alphaD56A) or beta subunit Lys167 (betaK167T), residues that interact in some crystal structures of the tryptophan synthase alpha2beta2 complex, decreases the ability of the alpha subunit to activate the beta subunit and alters the reaction and substrate specificity of the beta subunit. Partial conformational repair is provided by alpha-glycerol 3-phosphate, a ligand that binds to the alpha subunit, or by Cs+ or NH4+, ligands that bind to the beta subunit. Mutation of beta subunit Arg175 (betaR175A), a residue that interacts with alpha subunit Pro57 in some structures, has much smaller effects on activity but results in a 15-fold increase in the apparent Kd for dissociation of the alpha and beta subunits. Replacement of the single tryptophan in the beta subunit by phenylalanine (W177F) has only small effects on activity but increases the apparent subunit dissociation constant approximately 10-fold. The most important conclusions of this investigation are that interaction between alphaAsp56 and betaLys167 is important for intersubunit communication and that mutual stabilization of the active conformations of the two subunits is impaired by mutation of either residue.

Amino Acid Substitution↗
PubMed1998

The importance of employee demographic profiles for understanding experiences of work-family interrole conflicts.

This article investigated the reported experiences of work-family interrole conflict differences between American men and women. Participants answered questions on several questionnaires and were classified according to marital status, working or non-working spouse or partner, and presence of children. Overall, men reported significantly higher levels of work-to-family and strain-based, family-to-work conflict. Married men with working spouses, with and without children, and married men with children and non-working spouses reported higher levels of time-based and strain-based, family-to-work conflict than women in each corresponding category. The highest time-based, family-to-work conflict was reported by divorced women with children.

Conflict, Psychological↗
PubMed1997

Crystal structures of a mutant (betaK87T) tryptophan synthase alpha2beta2 complex with ligands bound to the active sites of the alpha- and beta-subunits reveal ligand-induced conformational changes.

Three-dimensional structures are reported for a mutant (betaK87T) tryptophan synthase alpha2beta2 complex with either the substrate L-serine (betaK87T-Ser) or product L-tryptophan (betaK87T-Trp) at the active site of the beta-subunit, in which both amino acids form external aldimines with the coenzyme, pyridoxal phosphate. We also present structures with L-serine bound to the beta site and either alpha-glycerol 3-phosphate (betaK87T-Ser-GP) or indole-3-propanol phosphate (betaK87T-Ser-IPP) bound to the active site of the alpha-subunit. The results further identify the substrate and product binding sites in each subunit and provide insight into conformational changes that occur upon formation of these complexes. The two structures having ligands at the active sites of both alpha- and beta-subunits reveal an important new feature, the ordering of alpha-subunit loop 6 (residues 179-187). Closure of loop 6 isolates the active site of the alpha-subunit from solvent and results in interaction between alphaThr183 and the catalytic residue alphaAsp60. Other conformational differences between the wild type and these two mutant structures include a rigid-body rotation of the alpha-subunit of approximately 5 degrees relative to the beta-subunit and large movements of part of the beta-subunit (residues 93-189) toward the rest of the beta-subunit. Much smaller differences are observed in the betaK87T-Ser structure. Remarkably, binding of tryptophan to the beta active site results in conformational changes very similar to those observed in the betaK87T-Ser-GP and betaK87T-Ser-IPP structures, with exception of the disordered alpha-subunit loop 6. These large-scale changes, the closure of loop 6, and the movements of a small number of side chains in the alpha-beta interaction site provide a structural base for interpreting the allosteric properties of tryptophan synthase.

Binding Sites↗
PubMed1997

Importance of conserved and variable C-terminal residues for the activity and thermal stability of the beta subunit of tryptophan synthase.

To assess the functional roles of helix 13 and of the conserved and variable residues in the C-terminal region (residues 378-397) of the tryptophan synthase beta subunit, we have constructed four C-terminal truncations and 12 point mutations. The effects of these mutations on kinetic and spectroscopic properties and thermal stability are reported here. The mutant beta subunits all form stable alpha2beta2 complexes that have been purified to homogeneity. The mutant alpha2beta2 complexes are divided into two classes on the basis of activity in the reaction of L-serine with indole to form tryptophan. Class I enzymes, which have mutations at Arg-379 or Asp-381 or truncations (384-397 or 385-397), exhibit significant activity (1-38% of wild type). Class II enzymes, which have mutations at Lys-382 or Asp-383 or truncations (382-397 or 383-397), exhibit very low activity (<1% of wild type). Although Class II enzymes have drastically reduced activity in the reaction of L-serine with indole and an altered distribution of enzyme-substrate intermediates in the reaction of L-serine with beta-mercaptoethanol, they retain activity in the reaction of beta-chloro-L-alanine with indole. Correlation of the results with the three-dimensional structure of the alpha2beta2 complex suggests that Lys-382 and Asp-383 serve important roles in a proposed "open" to "closed" conformational change that occurs in the reactions of L-serine. Because mutant beta subunits having C-terminal truncations (383-397 or 384-397) undergo much more rapid thermal inactivation at 60 degrees C than the wild type beta subunit, the C-terminal helix 13 stabilizes the beta subunit.

Amino Acid Sequence↗
PubMed1997

Dissociation equilibria of the tryptophan synthase alpha 2 beta 2 complex in saline buffer and guanidine isothiocyanate, as studied by sedimentation equilibrium.

The dissociation equilibria of Salmonella typhimurium tryptophan synthase alpha 2 beta 2 complex were studied via centrifugation of the complex to sedimentation equilibrium in neutral saline buffers containing 0 to 137 mM guanidine isothiocyanate (GuSCN). The resulting concentration gradients were analyzed in the context of an equilibrium model for sequential dissociation of two alpha subunits from a stable beta 2 subunit. Under the conditions of these experiments, the first dissociation constant alone could be evaluated at GuSCN concentrations < or = 100 mM, and the second dissociation constant alone could be evaluated at GuSCN = 137 mM. At intermediate GuSCN, both dissociation constants were sufficiently well defined to rule out the presence of a large equilibrium cooperative effect in the stepwise dissociation of the alpha subunits.

Bacterial Proteins↗
PubMed1996

Mechanism of activation of the tryptophan synthase alpha2beta2 complex. Solvent effects of the co-substrate beta-mercaptoethanol.

To characterize the conformational transitions that lead to activation of catalysis by the tryptophan synthase alpha2beta2 complex, we have determined the solvent effects of a co-substrate, beta-mercaptoethanol, and of a model nonsubstrate, ethanol, on the catalytic and spectroscopic properties of the enzyme. Our results show that ethanol and beta-mercaptoethanol both alter the equilibrium distribution of pyridoxal 5'-phosphate intermediates formed in the reactions of L-serine at the beta site in the alpha2beta2 complex. Addition of increasing concentrations of ethanol increases the proportion of the external aldimine of L-serine and decreases the proportion of the external aldimine of aminoacrylate. Low concentrations of the co-substrate beta-mercaptoethanol (Kd = approximately 13 mM) decrease the proportion of the external aldimine of aminoacrylate and induce formation of the quinonoid of S-hydroxyethyl-L-cysteine. Higher concentrations of beta-mercaptoethanol decrease the concentration of the quinonoid intermediate and increase the proportion of the external aldimine of L-serine. Data analysis shows that beta-mercaptoethanol and ethanol both interact or bind preferentially with the conformer of the enzyme that predominates when the aldimine of L-serine is formed and shift the equilibrium in favor of this conformer. We propose that a nonpolar region of the beta subunit, possibly the hydrophobic indole tunnel, becomes less exposed to solvent in the conformational transition that activates the alpha2beta2 complex.

Allosteric Regulation↗
PubMed1996

A thermally induced reversible conformational transition of the tryptophan synthase beta2 subunit probed by the spectroscopic properties of pyridoxal phosphate and by enzymatic activity.

A reversible thermally induced conformational transition of the beta2 subunit of tryptophan synthase from Salmonella typhimurium has been detected by use of the pyridoxal 5'-phosphate coenzyme as a spectroscopic probe. Increasing the temperature converts the major form of pyridoxal 5'-phosphate bound to the beta2 subunit from a ketoenamine species with lambdamax at 410 nm to a enolimine species with lambdamax at 336 nm (Tm = approximately 43 degrees C) and results in loss of the circular dichroism signal at 410 nm and of fluorescence emission at 510 nm. The results indicate that increasing the temperature favors a conformer of the enzyme that binds pyridoxal 5'-phosphate in a more nonpolar environment and leads to loss of asymmetric pyridoxal 5'-phosphate binding. The internal aldimine between pyridoxal 5'-phosphate and the epsilon-amino group of lysine 87 is not disrupted by increased temperature because sodium borohydride treatment of the enzyme at either 15 or 60 degrees C results in covalent attachment of [4'-3H]pyridoxal 5'-phosphate. The thermal transition of the beta2 subunit below 60 degrees C produces reversible thermal inactivation (Ti = approximately 52 degrees C) and occurs at a much lower temperature than the major reversible unfolding at approximately 80 degrees C (Remeta, D. P., Miles, E. W., and Ginsburg, A. (1995) Pure Appl. Chem. 67, 1859-1866). Our new results indicate that the 410 nm absorbing species of pyridoxal 5'-phosphate is the catalytically active form of the cofactor in the beta2 subunit and that the low temperature reversible conformational transition disturbs the active site and causes loss of catalytic activity.

Borohydrides↗
PubMed1996

Exchange of K+ or Cs+ for Na+ induces local and long-range changes in the three-dimensional structure of the tryptophan synthase alpha2beta2 complex.

Monovalent cations activate the pyridoxal phosphate-dependent reactions of tryptophan synthase and affect intersubunit communication in the alpha2beta2 complex. We report refined crystal structures of the tryptophan synthase alpha2beta2 complex from Salmonella typhimurium in the presence of K+ at 2.0 angstrom and of Cs+ at 2.3 angstrom. Comparison of these structures with the recently refined structure in the presence of Na+ shows that each monovalent cation binds at approximately the same position about 8 angstrom from the phosphate of pyridoxal phosphate. Na+ and K+ are coordinated to the carbonyl oxygens of beta Phe-306, beta Ser-308, and beta Gly-232 and to two or one water molecule, respectively. Cs+ is coordinated to the carbonyl oxygens of beta Phe-306, beta Ser-308, beta Gly-232, beta Val-231, beta Gly-268 and beta Leu-304. A second binding site for Cs+ is located in the beta/beta interface on the 2-fold axis with four carbonyl oxygens in the coordination sphere. In addition to local changes in structure close to the cation binding site, a number of long-range changes are observed. The K+ and Cs+ structures differ from the Na+ structure with respect to the positions of beta Asp-305, beta Lys-167, and alpha Asp-56. One unexpected result of this investigation is the movement of the side chains of beta Phe-280 and beta Tyr-279 from a position partially blocking the tunnel in the Na+ structure to a position lining the surface of the tunnel in the K+ and Cs+ structures. The results provide a structural basis for understanding the effects of cations on activity and intersubunit communication.

Amino Acid Sequence↗
PubMed1996

Allosteric regulation of tryptophan synthase: effects of pH, temperature, and alpha-subunit ligands on the equilibrium distribution of pyridoxal 5'-phosphate-L-serine intermediates.

The equilibrium distribution of catalytic intermediates formed in the reaction of L-serine with the tryptophan synthase alpha 2 beta 2-complex from Salmonella typhimurium has been investigated by absorption and fluorescence spectroscopy as a function of pH, temperature, and alpha-subunit ligands. The novel result of this study is that the equilibrium between the two major catalytic species, the external aldimine and the alpha-aminoacrylate, is modulated by the ionization of two groups with apparent pK values of 7.8 +/- 0.3 and 10.3 +/- 0.2. Protonation of these groups stabilizes the alpha-aminoacrylate Schiff base by an estimated 100-fold with respect to the external aldimine. Furthermore, the formation of the alpha-aminoacrylate from the external aldimine is an endothermic process. Temperature slightly affects the apparent pK values but remarkably influences the amplitude of the phase associated with the ionization of each group. At 20 degrees C, each phase accounts for nearly half of the titration. Since the isolated beta 2-dimer does not exhibit a pH-dependent distribution of intermediates, the alpha-beta-subunit interactions seem critical to the onset of this functional property of the beta-subunit. The modulation of intersubunit interactions by the alpha-subunit ligands DL-alpha-glycerol 3-phosphate and phosphate leads to significant changes in the pH-dependent distribution of intermediates. At saturating concentrations of either of these alpha-subunit ligands, the alpha-aminoacrylate Schiff base is the predominant species over a wide pH range while the apparent pK values of the groups that control the equilibrium are not significantly affected. The pH-dependent interconversion of catalytic intermediates here reported has not been previously detected because phosphate buffers have usually been employed in the studies of this enzyme. Our findings are discussed in the light of a model in which specific protein conformations are associated with the external aldimine and the alpha-aminoacrylate Schiff bases, the latter being stabilized by temperature, protons, and alpha-subunit ligands.

Allosteric Regulation↗
PubMed1996

PCR mutagenesis and overexpression of tryptophan synthase from Salmonella typhimurium: on the roles of beta2 subunit Lys-382.

We have devised convenient methods for mutagenesis and very high level expression of wild type and mutant tryptophan synthase alpha and beta2 subunits and alpha2beta2 complex from Salmonella typhimurium. The trpBA genes were modified by introduction of five new restriction sites by polymerase chain reaction (PCR) and were then cloned into the plasmid pTrc99A under trc promoter control. The recombinant plasmid pEBA-10 and three plasmids constructed from pEBA-10 were transformed into Escherichia coli CB149, which lacks tryptophan operon genes. Optimization of growth conditions of the transformed cells resulted in 10- to 40-fold higher yields of cells ( approximately 22 g/liter) than attained previously. The improved expression system gave higher yields of tryptophan synthase proteins (23-70% of the soluble protein) and led to correspondingly high yields of purified alpha and beta2 subunits or alpha2beta2 complex (200-800 mg/liter). A plasmid containing 8 copies of the trpA gene gave the highest yield of alpha subunit. The PCR-based mutagenesis method permits mutation of any base pair in the trpBA genes, between suitable pairs of restriction sites, and requires only one new primer per mutation. The method is illustrated by construction of mutant beta2 subunits with any of five amino acid substitutions at Lys-382, the site of a previously described missense mutation. Characterization of the purified mutant alpha2beta2 complexes shows that Lys-382 in the wild type alpha2beta2 complex does not serve an essential catalytic role but may stabilize an active "closed" conformation of the enzyme by forming a salt bridge with Glu-350.

Bacterial Proteins↗
PubMed1995

Kinetic characterization of channel impaired mutants of tryptophan synthase.

Tryptophan synthase, an alpha 2 beta 2 tetrameric complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. The solution of the three-dimensional structure of the enzyme from Salmonella typhimurium provided physical evidence for a 25-A hydrophobic tunnel which connects the alpha and beta active sites (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). Using rapid reaction kinetics, we have previously established that indole is indeed channeled and have identified three essential kinetic features which govern efficient channeling. In the current study we have probed the necessity of these features by using site-directed mutagenesis to alter these requirements. We now report the kinetic characterization of two mutants which contain substitutions to block or restrict the tunnel (beta C170F and beta C170W). Preliminary kinetic and structural evidence of a restricted tunnel in the beta C170W has been provided (Schlichting, I., Yang, X. W., Miles, E. W., Kim, A. Y., and Anderson, K. S. (1994) J. Biol. Chem. 269, 26591-26593). The rapid kinetic analysis of these mutant proteins shows that these mutations interfere with efficient channeling of the indole metabolite such that indole can be observed in single enzyme turnover of the physiologically relevant alpha beta reaction. In addition, the beta C170W mutant appears to be impaired in alpha beta intersubunit communication.

Binding Sites↗
PubMed1995

The tryptophan synthase alpha 2 beta 2 complex: kinetic studies with a mutant enzyme (beta K87T) to provide evidence for allosteric activation by an aminoacrylate intermediate.

To investigate the mechanism by which the tryptophan synthase beta subunit accelerates the cleavage of the indole-3-glycerol phosphate catalyzed by the alpha subunit (alpha reaction), kinetic experiments were carried out with wild-type and mutant forms of the alpha 2 beta 2 complex. Previous studies indicate that this activation can be attributed to the conformational changes associated with the formation of a Schiff base between aminoacrylate and pyridoxal phosphate at the beta site. To test this hypothesis, we investigated a mutant form of the alpha 2 beta 2 complex having the lysine-87 of its beta subunits replaced by threonine. The mutant alpha 2 beta 2 complex (K87T) exhibits normal activity for the alpha reaction but fails to catalyze formation of L-tryptophan from L-serine and indole (beta reaction). However, the mutant enzyme can form a Schiff base intermediate with L-serine at the beta site. Using a "chemical rescue" method, we converted K87T L-serine intermediate to an aminoacrylate intermediate. Steady-state kinetic studies reveal that the aminoacrylate derivative exhibits a 7-fold enhancement in kcat/Km for the alpha reaction relative to that of the L-serine derivative of the mutant or the wild-type enzyme in the absence of L-serine. Rapid kinetic data show that the aminoacrylate derivative of the mutant enzyme exhibits a 6-fold increase in the rate constant for the indole-3-glycerol phosphate cleavage reaction. In addition, rate constants for the reverse reaction and product release steps are also altered. Together, these changes lead to a decrease in Km and an increase in kcat.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylates↗
PubMed1995

Monovalent cations partially repair a conformational defect in a mutant tryptophan synthase alpha 2 beta 2 complex (beta-E109A).

We are using the tryptophan synthase alpha 2 beta 2 complex as a model system to investigate how ligands, protein-protein interaction, and mutations regulate enzyme activity, reaction specificity, and substrate specificity. The rate of conversion of L-serine and indole to L-tryptophan by the beta 2 subunit alone is quite low, but is activated by certain monovalent cations or by association with alpha subunit to form an alpha 2 beta 2 complex. Since monovalent cations and alpha subunit appear to stabilize an active conformation of the beta 2 subunit, we have investigated the effects of monovalent cations on the activities and spectroscopic properties of a mutant form of alpha 2 beta 2 complex having beta 2 subunit glutamic acid 109 replaced by alanine (E109A). The E109A alpha 2 beta 2 complex is inactive in reactions with L-serine but active in reactions with beta-chloro-L-alanine. Parallel experiments show effects of monovalent cations on the properties of wild type beta 2 subunit and alpha 2 beta 2 complex. We find that CsCl stimulates the activity of the E109A alpha 2 beta 2 complex and of wild type beta 2 subunit with L-serine and indole and alters the equilibrium distribution of L-serine reaction intermediates. The results indicate that CsCl partially repairs the deleterious effects of the E109A mutation on the activity of the alpha 2 beta 2 complex by stabilizing a conformation with catalytic properties more similar to those of the wild type alpha 2 beta 2 complex. This conclusion is consistent with observations that monovalent cations alter the catalytic and spectroscopic properties of several pyridoxal phosphate-dependent enzymes by stabilizing alternative conformations.

Binding Sites↗
PubMed1995

Subunit assembly in the tryptophan synthase alpha 2 beta 2 complex. Stabilization by pyridoxal phosphate aldimine intermediates.

This work is aimed at understanding subunit assembly in the tryptophan synthase alpha 2 beta 2 complex and the importance of the internal aldimine between pyridoxal phosphate and lysine 87 of the beta 2 subunit of tryptophan synthase for subunit association. We utilize a mutant form of the beta 2 subunit that is unable to form the internal aldimine because lysine 87 is replaced by threonine (K87T). The K87T alpha 2 beta 2 complex is inactive in reactions catalyzed by the beta 2 subunit but retains activity in the reaction catalyzed by the alpha subunit. We find that dialysis removes pyridoxal phosphate much more rapidly from the K87T beta 2 subunit and alpha 2 beta 2 complex than from the wild type counterparts. Activity measurements, gel filtration, and subunit interchange experiments show that the alpha subunit dissociates more readily from the K87T beta 2 subunit than from the wild type beta 2 subunit. The reaction of L-serine to form an external aldimine with pyridoxal phosphate at the active site of the K87T beta 2 subunit markedly increases the affinity for the alpha subunit and slows removal of pyridoxal phosphate by dialysis. We propose that the external aldimine between L-serine and pyridoxal phosphate bridges the N-domain and the C-domain in the K87T beta 2 subunit. This interdomain bridge may mimic the internal aldimine bond in the wild type beta 2 subunit and stabilize pyridoxal phosphate binding. The interdomain bridges formed by the internal aldimine with the wild type beta 2 subunit and by the external aldimine with L-serine in the K87T beta 2 subunit may further stabilize interaction with the alpha subunit because the alpha/beta interaction site contains residues from both N- and C-domains of the beta 2 subunit.

Chromatography, Gel↗
PubMed1995

Ligand-mediated changes in the tryptophan synthase indole tunnel probed by nile red fluorescence with wild type, mutant, and chemically modified enzymes.

The bacterial tryptophan synthase alpha 2 beta 2 complex contains an unusual structural feature: an intramolecular tunnel that channels indole from the active site of the alpha subunit to the active site of the beta subunit 25 A away. Here we investigate the role of the tunnel in communication between the alpha and beta subunits using the polarity-sensitive fluorescent probe, Nile Red. Interaction of Nile Red in the nonpolar tunnel near beta subunit residues Cys-170 and Phe-280 is supported by studies with enzymes altered at these positions. Restricting the tunnel by enlarging Cys-170 by chemical modification or mutagenesis decreases the fluorescence of Nile Red by 30-70%. Removal of a partial restriction in the tunnel by replacing Phe-280 by Cys or Ser increases the fluorescence of Nile Red more than 2-fold. A binding site for Nile Red in this region near the pyridoxal phosphate coenzyme of the beta subunit is further supported by iodide quenching and fluorescence energy transfer experiments and by molecular modeling based on the three-dimensional structure of the alpha 2 beta 2 complex. Finally, studies using Nile Red as a sensitive probe of conformational changes in the tunnel reveal that allosteric ligands (alpha subunit) or active site ligands (beta subunit) decrease the fluorescence of Nile Red. We speculate that allosteric and active site ligands induce a tunnel restriction near Phe-280 that serves as a gate to control passage of indole through the tunnel.

Amino Acid Sequence↗
PubMed1994

Structural and kinetic analysis of a channel-impaired mutant of tryptophan synthase.

Substrate channeling is a process by which two sequential enzymes interact to transfer a metabolite (or intermediate) from one enzyme active site to the next without allowing free diffusion of the metabolite. Channeling is thought to play an important role in metabolic regulation and cellular modulation of enzymatic activities. Although there are numerous examples of sequential enzyme pairs in glycolysis and other biosynthetic pathways that are thought to exhibit channeling, this is as yet controversial. Tryptophan synthase is considered the best example for an enzyme displaying channeling behavior between subunits. Tryptophan synthase is an alpha 2 beta 2 tetrameric enzyme complex, which catalyzes the last two steps in the biosynthesis of L-tryptophan. The alpha subunit catalyzes the cleavage of indole-3-glycerol phosphate to indole and glyceraldehyde-3-phosphate; the beta subunit catalyzes the condensation of indole with serine to form tryptophan, in a reaction mediated by pyridoxal phosphate. The inability to trap free indole in the steady-state reaction and analysis of the kinetics of single turnover reactions have led to the postulate that indole may pass directly from the alpha to the beta site without diffusion through solution (Demoss, J. A. (1962) Biochim. Biophys. Acta 62, 279-293; Matchett, W. M. (1974) J. Biol. Chem. 249, 4041-4049). The crystal structure of tryptophan synthase from Salmonella typhimurium has provided additional support for substrate channeling by elucidating a 25-A hydrophobic tunnel connecting the two catalytic sites (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). The structure suggests that mutation of a residue lining the tunnel to a more bulky residue might impede or block the passage of indole during catalysis thus enabling detection of indole during a single enzyme turnover. A mutant of tryptophan synthase has been prepared in which one of the residues lining the tunnel, beta Cys-170, has been replaced with a bulkier tryptophan residue. Kinetic and structural analyses of the beta C170W mutant by rapid chemical quench methods and x-ray crystallographic analysis show both the transient formation of indole and the obstruction of the tunnel, thus providing direct evidence for the substrate channeling mechanism.

Catalysis↗