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The Tryptophan Synthase α2β2 Complex: A Model for Substrate Channeling, Allosteric Communication, and Pyridoxal Phosphate Catalysis

Open AccessPublished:February 20, 2013DOI:https://doi.org/10.1074/jbc.X113.463331
      I reflect on my research on pyridoxal phosphate (PLP) enzymes over fifty-five years and on how I combined research with marriage and family. My Ph.D. research with Esmond E. Snell established one aspect of PLP enzyme mechanism. My postdoctoral work first with Hans L. Kornberg and then with Alton Meister characterized the structure and function of another PLP enzyme, l-aspartate β-decarboxylase. My independent research at the National Institutes of Health (NIH) since 1966 has focused on the bacterial tryptophan synthase α2β2 complex. The β subunit catalyzes a number of PLP-dependent reactions. We have characterized these reactions and the allosteric effects of the α subunit. We also used chemical modification to probe enzyme structure and function. Our crystallization of the tryptophan synthase α2β2 complex from Salmonella typhimurium led to the determination of the three-dimensional structure with Craig Hyde and David Davies at NIH in 1988. This landmark structure was the first structure of a multienzyme complex and the first structure revealing an intramolecular tunnel. The structure has provided a basis for exploring mechanisms of catalysis, channeling, and allosteric communication in the tryptophan synthase α2β2 complex. The structure serves as a model for many other multiprotein complexes that are important for biological processes in prokaryotes and eukaryotes.

      B.A. at the University of Texas at Austin (1954–1957)

      I attended the University of Texas in Austin (UT-Austin), where my father was an associate professor of English. My mother was a librarian. My parents encouraged me to study hard and to pursue any career that interested me. Although I started at UT-Austin as a premed major, I changed to a chemistry major after working as a technician in a biochemistry laboratory. I enjoyed experimental work and contacts with graduate students. I heard good things about Esmond Snell, who was a professor of chemistry at UT and was to go to University of California, Berkeley (UC-Berkeley), in 1956 to be chairman of the Department of Biochemistry. Snell's reputation as an outstanding biochemist was one factor in my decision to start graduate work at UC-Berkeley in 1957.

      Ph.D. at UC-Berkeley (1957–1961)

      The staff and students in the Department of Biochemistry at UC-Berkeley welcomed me warmly. I learned basic biochemistry in courses taught by Esmond Snell and Frederick Carpenter. The second year, I studied basic enzymology and took an enzyme lab course taught by Jesse Rabinowitz. I decided to do my doctoral research under Esmond Snell with Jesse Rabinowitz and Edward Adelberg as thesis advisors. Snell's outstanding work included the discovery of two new forms of vitamin B6, pyridoxal and pyridoxamine, and the elucidation of the general basis for catalysis by vitamin B6-dependent enzymes. My Ph.D. thesis work was designed to test one aspect of Snell's proposed general mechanism that differed from a similar mechanism proposed independently by A. E. Braunstein (Fig. 1). I isolated bacteria from Strawberry Creek on the UC campus that used α-methylserine as a sole source of carbon and nitrogen. From these bacteria, I partially purified and characterized a pyridoxal phosphate (PLP)- and tetrahydrofolate-dependent α-methylserine hydroxymethyltransferase. The enzyme catalyzed the reversible cleavage of α-methylserine to d-alanine and formaldehyde. My results showed that this reaction does not require labilization of an α-hydrogen (
      • Wilson E.M.
      • Snell E.E.
      Metabolism of α-methylserine. I. α-Methylserine hydroxymethyltransferase.
      ,
      • Wilson E.M.
      • Snell E.E.
      Metabolism of α-methylserine. II. Stereospecificity of α-methylserine hydroxymethyltransferase.
      ). Braunstein's mechanism did include labilization of an α-hydrogen. Drs. Beverly Guirard and Hiroshi Wada were important influences on me in the Snell laboratory (Fig. 2). Hiroshi Wada was the first of many Japanese scientists to study PLP enzymes with Snell. Jesse Rabinowitz helped me with the tetrahydrofolate coenzyme in my enzyme. I also learned about PLP enzymes from a new young faculty member, Terry Jenkins, who had recently characterized highly purified glutamic aspartatic aminotransferase for his Ph.D. research with Irwin Sizer at the Massachusetts Institute of Technology. The Department of Biochemistry had an excellent series of guest speakers on Friday afternoons. I was especially impressed by the lecture by Hans L. Kornberg, who had recently discovered the glyoxylate cycle, and I applied to do postdoctoral work in his laboratory.
      Figure thumbnail gr1
      FIGURE 1Edith Wilson uses a Cary recording spectrophotometer in the Department of Biochemistry at UC-Berkeley in 1958.
      Figure thumbnail gr2
      FIGURE 2Celebration of Esmond Snell's seventieth birthday at the American Society for Biochemistry and Molecular Biology (ASBMB) meeting in St. Louis in 1984. Left to right: Edith Miles, Gene Brown, Hiroshi Wada, Esmond Snell, and Mary Snell. This photograph was provided by Professor Sumio Tanase.

      Postdoctoral Work (1962–1966)

      My postdoctoral work with Hans Kornberg at the University of Leicester in England from 1962 through 1963 was funded by an American Cancer Society grant (Fig. 3). My problem was to understand the control of the glyoxylate cycle in Achromobacter d-15 and how the control differed from that of the glyoxylate cycle in Escherichia coli. I used isotopes in short-term growth experiments and measured enzymatic activities in cell-free extracts. Many of the assays and oxidation experiments utilized Warburg manometers. I joked that I was at the bottom of a line of succession: I learned to use the Warburg manometer from Hans Kornberg, who had learned from Hans Krebs, who had learned from Otto Warburg. I found an important difference between Achromobacter d-15 and E. coli. Achromobacter d-15 lacked oxaloacetate decarboxylase but had high levels of l-aspartate 4-carboxy-lyase, a PLP-dependent enzyme that converts l-aspartate to α-alanine and carbon dioxide. I purified this PLP enzyme and characterized a number of its properties (
      • Wilson E.M.
      • Kornberg H.L.
      Properties of crystalline l-aspartate 4-carboxy-lyase from Achromobacter sp.
      ). I obtained beautiful hexagonal crystals of the purified enzyme that were too thin for x-ray crystallography at that time. The most important thing I learned was that my main love was the study of pure PLP enzymes, not the study of metabolic control.
      Figure thumbnail gr3
      FIGURE 3Sir Hans Kornberg, FRS. This photograph was taken by Jason Varney.
      I enjoyed a second postdoctoral position with Alton Meister in the Department of Biochemistry at Tufts University School of Medicine in Boston (Fig. 4). His group was studying l-aspartate 4-carboxy-lyase (also known as l-aspartate β-decarboxylase) from a different source, Alcaligenes faecalis. To learn more about the binding of PLP to this enzyme, I determined the optical rotatory dispersion of various forms of the enzyme and compared the Cotton effects obtained with those that had been reported for glutamate-aspartate transaminase (
      • Wilson E.M.
      • Meister A.
      Optical rotatory dispersion of l-aspartate β-decarboxylase and its derivatives.
      ). I next studied the reaction of β-hydroxyaspartate with l-aspartate 4-carboxy-lyase and postulated a new mechanism of inhibition (
      • Miles E.W.
      • Meister A.
      The mechanism of the reaction of β-hydroxyaspartate with l-aspartate β-decarboxylase. A new type of pyridoxal 5′-phosphate-enzyme inhibition.
      ). I made many friends and important professional contacts at Tufts, including Kenji Soda, James Manning, Jonathan Nishimura, Paul Anderson, Elizabeth Mooz, Mahtab Bamji, and Vaira and Daniel Wellner.
      Figure thumbnail gr4
      FIGURE 4Alton Meister. This photograph was supplied by ASBMB.

      Independent Research at the National Institutes of Health (1966–Present)

      I was fortunate to be offered an independent position in the Laboratory of Biochemical Pharmacology at the National Institute of Arthritis and Metabolic Diseases (NIAMD) of the National Institutes of Health (NIH) in 1966. I have spent the rest of my career at NIH in the same laboratory, which has been renamed the Laboratory of Biochemistry and Genetics. I have been a Scientist Emeritus since 2000.
      My studies of PLP enzymes during my doctoral and postdoctoral work convinced me that PLP enzymes are ideal targets for studies of enzyme structure and function. The PLP coenzyme provides a spectrophotometric probe that is useful for following catalytic intermediates, reaction kinetics, and conformational changes. My search for an especially interesting PLP enzyme for my independent work led me to tryptophan synthase. Irving Crawford and Charles Yanofsky had reported that E. coli tryptophan synthase is an enzyme complex consisting of two separable polypeptides, now designated the α subunit and the β subunit (
      • Crawford I.P
      • Yanofsky C.
      On the separation of the tryptophan synthetase of Escherichia coli into two protein components.
      ). The separate α subunit catalyzes the α reaction (Equation 1).
      Indole-3-glycerolphosphateindole+D-glyceraldehyde3-phosphate
      (Eq. 1)


      The separate β subunit catalyzes the PLP-dependent β reaction (Equation 2).
      Indole+L-serineL-tryptophan+H2O
      (Eq. 2)


      The activity of each separate enzyme is increased by 30–100-fold when one subunit is mixed with the other subunit. The physiologically important reaction, the αβ reaction (Equation 3), is catalyzed only by the α2β2 complex.
      Indole-3-glycerolphosphate+L-serineL-tryptophan+D-glyceraldehyde3-phosphate+H2O
      (Eq. 3)


      The αβ reaction is essentially the sum of the α reaction and the β reaction. Indole is an intermediate in the αβ reaction. The finding that the reaction rates of the β subunit in the β reaction were increased by 30-fold by interaction with the α subunit suggested that this would be a good system for investigating the specificity and control of the catalytic site of a PLP enzyme.
      I started my work at NIH with generous help from Irving Crawford at the Scripps Clinic and Research Foundation in La Jolla, California. Irving sent me purified enzymes and E. coli strains for preparing the separate α and β subunits. Absorption spectra of the β subunit in the presence of l-serine showed that the β subunit converted l-serine to pyruvate (Equation 4).
      L-Serinepyruvae+ammonia
      (Eq. 4)


      The absorption spectra of the β subunit in the presence of l-serine and mercaptoethanol suggested that PLP was converted to pyridoxamine phosphate (PMP) by a transamination reaction. I learned from Irving Crawford that he and M. Hatanaka had earlier found evidence for a transamination reaction. However, they had been unable to publish their results because they had not identified the keto acid reaction product. Irving and I decided to collaborate on this problem. Fortunately, I was able to work with him in his laboratory at the Scripps Clinic and Research Foundation for three weeks in the summer of 1967 on my way to the International Congress of Biochemistry in Tokyo. My husband, Harry Todd Miles, worked with Leslie Orgel during the same time. This was a very productive three weeks for all of us. Irving and I carried out many essential experiments for a joint paper (
      • Miles E.W.
      • Hatanaka M.
      • Crawford I.P.
      A new thiol-dependent transamination reaction catalyzed by the B protein of Escherichia coli tryptophan synthetase.
      ). We demonstrated that the β subunit converted l-serine and mercaptoethanol to S-hydroxyethyl-l-cysteine (Equation 5), as demonstrated previously for the α2β2 complex.
      Mercaptoethanol+L-serineS-hydroxyethyl-L-cysteine+H2O
      (Eq. 5)


      We identified S-pyruvylmercaptoethanol and PMP as the products of the new transamination reaction (Equation 6).
      PLP+L-serine+mercaptoethanolS-pyruvylmercaptoethanol+PMP+H2O
      (Eq. 6)


      This transamination reaction is useful for removing PLP from the β subunit to give the apo-β subunit.
      The results in the article showed that the α subunit completely inhibits the serine deaminase reaction (Equation 4) and the transaminase reaction (Equation 6) but stimulates the β replacement reactions (Equations 2 and 5). Thus, tryptophan synthase is a promising system for studying how subunit interaction controls reaction rates and reaction specificity in a multienzyme complex.
      Irving Crawford was an important mentor to me and helped me to get my research at NIH off to a good start. Although we never published another paper together, we had extensive correspondence and enjoyed meeting each other at tryptophan conferences. Unfortunately, he died prematurely in 1989.

      Chemical Modification

      In some of my early work at NIH, I used chemical modification to identify key residues in the β subunit and to determine the effects of modification on enzymatic activities and subunit interaction. I found that 5,5′-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide modify two SH residues in the apo-β subunit and one SH residue in the holo-β subunit. Interestingly, modification of the holo-β subunit by N-ethylmaleimide stimulates activity in Equation 4 but inhibits activity in Equation 2 (
      • Miles E.W.
      The B protein of Escherichia coli tryptophan synthetase. I. Effects of sulfhydryl modification on enzymatic activities and subunit interaction.
      ). We later modified an essential histidyl residue in the β subunit by photo-oxidation in the presence of PLP and l-serine and by reaction with diethylpyrocarbonate (
      • Miles E.W.
      • Kumagai H.
      Modification of essential histidyl residues of the β2 subunit of tryptophan synthetase by photo-oxidation in the presence of pyridoxal 5′-phosphate and l-serine and by diethylpyrocarbonate.
      ). After this paper was published, I was invited to write a review for Methods in Enzymology on the modification of histidyl residues by diethylpyrocarbonate (
      • Miles E.W.
      Modification of histidyl residues in proteins by diethylpyrocarbonate.
      ). This review has been highly cited. We later modified arginine and amino groups.

      Proteolysis and Protein Folding

      We discovered that limited proteolysis of the α2β2 complex by trypsin produced a single site of cleavage in the α subunit at Arg-188 and produced an active “nicked” α2β2 complex (
      • Higgins W.
      • Fairwell T.
      • Miles E.W.
      An active proteolytic derivative of the α subunit of tryptophan synthase. Identification of the site of cleavage and characterization of the fragments.
      ). This report led to a productive collaboration with Katsuhide Yutani at the Institute for Protein Research of Osaka University in Japan. We found that guanidine hydrochloride induced stepwise unfolding of the α subunit. Parallel unfolding experiments with the two α subunit fragments provided evidence for the stepwise unfolding of the two α subunit domains (
      • Miles E.W.
      • Yutani K.
      • Ogasahara K.
      Guanidine hydrochloride induced unfolding of the α subunit of tryptophan synthase and of the two α proteolytic fragments: evidence for stepwise unfolding of the two α domains.
      ).

      New Reactions of Tryptophan Synthase and Reaction Mechanism

      Hidehiko Kumagai determined the activities of the tryptophan synthase β subunit and the tryptophan synthase α2β2 complex in β elimination reactions and β replacement reactions with different amino acid substrates. The results provide evidence that the reactions proceed through a key common enzyme-bound aminoacrylic intermediate, as reported previously for the reactions of tryptophanase with the same substrates. The main difference between the reactions of tryptophan synthase and tryptophanase is the failure of tryptophan synthase to catalyze the formation of pyruvate and indole from l-tryptophan (
      • Kumagai H.
      • Miles E.W.
      The B protein of Escherichia coli tryptophan synthetase. II. New β-elimination and β-replacement reactions.
      ). Later, Robert Phillips and I compared the inhibition of the tryptophan synthase α2β2 complex and tryptophanase by the tryptophan analogs oxindolyl-l-alanine and 2,3-dihydro-l-tryptophan, which have structures similar to the indolenine tautomer of l-tryptophan. Both compounds are very good competitive inhibitors of both enzymes. This result provides evidence that the indolenine tautomer of l-tryptophan is an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase (
      • Phillips R.S.
      • Miles E.W.
      • Cohen L.A.
      Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-l-alanine and 2,3-dihydro-l-tryptophan: support for an indolenine intermediate in tryptophan metabolism.
      ). Using fluorine NMR, we discovered two new, very slow reactions catalyzed by the tryptophan synthase α2β2 complex: the isomerization of 5-fluoro-l-tryptophan and (3S)-2,3-dihydro-5-fluoro-l-tryptophan (
      • Miles E.W.
      • Phillips R.S.
      • Yeh H.J.
      • Cohen L.A.
      Isomerization of (3S)-2,3-dihydro-5-fluoro-l-tryptophan and of 5-fluoro-l-tryptophan catalyzed by tryptophan synthase: studies using fluorine-19 nuclear magnetic resonance and difference spectroscopy.
      ).

      Enzyme Purification and Crystallization

      Early studies of E. coli tryptophan synthase showed that it was made up of two proteins, which were partially separated by chromatography on DEAE-cellulose (
      • Crawford I.P
      • Yanofsky C.
      On the separation of the tryptophan synthetase of Escherichia coli into two protein components.
      ). This was one of the first published uses of DEAE-cellulose. Subsequent studies of tryptophan synthase focused on the purification and characterization of the separate subunits from strains that contained only one of the two subunits. The α2β2 complex was then reconstituted from the isolated subunits. When Osao Adachi came to my laboratory, he wanted to purify the “native α2β2 complex” and to compare its properties with those of the α2β2 complex that was reconstituted from the isolated subunits. We were fortunate to receive from Charles Yanofsky a strain of E. coli that overproduced large amounts of both the α and β subunits. Adachi developed a five-step purification procedure that involved two chromatography steps on DEAE-Sephadex. Crystallization by ammonium sulfate in the final step yielded beautiful crystals with a uniform rod shape. The native and reconstituted α2β2 complexes exhibited identical absorption spectra, enzymatic activities, sedimentation velocity patterns, and bands on SDS gel electrophoresis (
      • Adachi O.
      • Kohn L.D.
      • Miles E.W.
      Crystalline α2β2 complexes of tryptophan synthetase of Escherichia coli. A comparison between the native complex and the reconstituted complex.
      ). Adachi also developed an improved procedure for preparing and crystallizing the β subunit (
      • Adachi O.
      • Miles E.W.
      A rapid method for preparing crystalline β2 subunit of tryptophan synthetase of Escherichia coli in high yield.
      ). Although we tried very hard to obtain crystals of the β subunit and α2β2 complex from E. coli that were large enough for x-ray crystallography, we did not succeed.
      Our later discovery that the Salmonella typhimurium α2β2 complex gives much better crystals than the E. coli enzyme is an example of serendipity. I was purifying the S. typhimurium enzyme for protein folding studies when I noticed that the enzyme crystallized in the fraction collection tubes. I suggested to a new postdoctoral fellow, Ashraf Ahmed, that he might try to grow crystals of the Salmonella enzyme for x-ray crystallography. After brief instructions on crystal growth from David Davies' laboratory, Ashraf grew excellent large crystals that diffracted well (
      • Ahmed S.A.
      • Miles E.W.
      • Davies D.R.
      Crystallization and preliminary x-ray crystallographic data of the tryptophan synthase α2β2 complex from Salmonella typhimurium.
      ). We demonstrated that the enzyme was fully functional in the crystalline state by studies with microcrystals (
      • Ahmed S.A.
      • Hyde C.C.
      • Thomas G.
      • Miles E.W.
      Microcrystals of tryptophan synthase α2β2 complex from Salmonella typhimurium are catalytically active.
      ). Microspectrophotometric studies on single crystals of the tryptophan synthase α2β2 complex demonstrated that substrates, substrate analogs, and reaction intermediate analogs form chromophoric intermediates with PLP at the active site of the β subunit (
      • Mozzarelli A.
      • Peracchi A.
      • Rossi G.L.
      • Ahmed S.A.
      • Miles E.W.
      Microspectrophotometric studies on single crystals of the tryptophan synthase α2β2 complex demonstrate formation of enzyme-substrate intermediates.
      ). Ligands that bind to the α subunit alter the distribution of intermediates at the β site in both the soluble and crystalline states.

      X-ray Crystallography

      The three-dimensional structure of the tryptophan synthase α2β2 complex from S. typhimurium revealed for the first time the architecture of a multienzyme complex and the presence of an intramolecular tunnel (Fig. 5) (
      • Hyde C.C.
      • Ahmed S.A.
      • Padlan E.A.
      • Miles E.W.
      • Davies D.R.
      Three-dimensional structure of the tryptophan synthase α2β2 multienzyme complex from Salmonella typhimurium.
      ). The four polypeptide chains are arranged in a nearly linear αββα order and form a complex 150 Å long. The active site of the α subunit was located by the presence of a substrate analog, indolepropanol phosphate. The active site of the β subunit was located by the presence of the PLP coenzyme. The active sites of the α and β subunits are 25 Å apart and are connected by a remarkable hydrophobic tunnel. The tunnel is believed to provide a passageway for the diffusion of indole from the site of its production from indole-3-glycerol phosphate at the active site of the α subunit to the site of tryptophan synthesis at the active site of the β subunit (see (Eq. 1), (Eq. 2), (Eq. 3)). Intramolecular tunneling could prevent the escape of indole to the solvent during catalysis. The structure showed that the α subunit has an 8-fold αβ barrel fold. The PLP coenzyme is sandwiched between the two domains of the β subunit. The β subunit structure was the first structure reported for an enzyme in a class of PLP enzymes termed group II. Other enzymes in this class include O-acetylserine sulfhydrylase, cystathionine β-synthase, and threonine dehydratase.
      Figure thumbnail gr5
      FIGURE 5Three-dimensional structure of the tryptophan synthase α2β2 multienzyme complex from S. typhimurium. The α subunits are in blue. β subunit N-terminal residues 1–204 and C-terminal residues 205–397 are shown in yellow and red, respectively. The dot surfaces highlight the positions of bound indolepropanol phosphate (red) in the active sites of the α subunit and the coenzyme PLP (dark blue) in the active sites of the β subunits. A tunnel that connects the two active sites (light blue) is shown in one αβ subunit pair. This figure was reprinted from Ref.
      • Hyde C.C.
      • Ahmed S.A.
      • Padlan E.A.
      • Miles E.W.
      • Davies D.R.
      Three-dimensional structure of the tryptophan synthase α2β2 multienzyme complex from Salmonella typhimurium.
      .
      Additional crystal structures have been determined on wild-type and mutant forms of the S. typhimurium tryptophan synthase α2β2 complex and in the presence of substrates and analogs by Sangkee Rhee in David Davies' group and by Ilme Schlichting's group. These structures demonstrate the presence of a series of PLP intermediates at the active site of the β subunit and in the presence and absence of ligands at the active site of the α subunit. The structural results, combined with kinetic and spectroscopic studies, provide evidence for allosteric communication and open and closed sites in the α and β subunits (see a recent review by Dunn (
      • Dunn M.F.
      Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex.
      )).

      Protein Engineering Identifies Key Residues

      While the initial crystal structure determination was under way, we developed methods of site-directed mutagenesis of tryptophan synthase from S. typhimurium. Our ability to make mutations in the S. typhimurium α2β2 complex would allow us to grow crystals of mutant forms of the S. typhimurium α2β2 complex as well as to characterize the mutant forms by spectroscopic and kinetic studies. After the structure was available, we mutated potentially important residues in the active sites of the α and β subunits and in the interaction site between the two subunits. Solution studies with nineteen mutants at α subunit Glu-49 showed that Glu-49 is essential for activity (
      • Miles E.W.
      • McPhie P.
      • Yutani K.
      Evidence that glutamic acid 49 of tryptophan synthase α subunit is a catalytic residue. Inactive mutant proteins substituted at position 49 bind ligands and transmit ligand-dependent effects to the β subunit.
      ). Asp-60 is another catalytic residue in the α subunit (
      • Nagata S.
      • Hyde C.C.
      • Miles E.W.
      The α subunit of tryptophan synthase. Evidence that aspartic acid 60 is a catalytic residue and that the double alteration of residues 175 and 211 in a second-site revertant restores the proper geometry of the substrate binding site.
      ). Cryocrystallography of the α2β2 complex with an α subunit D60N mutation with the true substrate revealed the correct orientation of α subunit active site Glu-49 (
      • Rhee S.
      • Miles E.W.
      • Mozzarelli A.
      • Davies D.R.
      Cryocrystallography and microspectrophotometry of a mutant (αD60N) tryptophan synthase α2β2 complex reveals allosteric roles of αAsp60.
      ).
      Mutations of the β subunit clarified the roles of β subunit His-86, Lys-87, Arg-148, Cys-170, and Cys-230 (
      • Miles E.W.
      • Kawasaki H.
      • Ahmed S.A.
      • Morita H.
      • Morita H.
      • Nagata S.
      The β subunit of tryptophan synthase. Clarification of the roles of histidine 86, lysine 87, arginine 148, cysteine 170, and cysteine 230.
      ). Crystal structures of the β subunit K87T mutant with ligands bound to the active sites of the α and β subunits revealed ligand-induced conformational changes (
      • Rhee S.
      • Parris K.D.
      • Hyde C.C.
      • Ahmed S.A.
      • Miles E.W.
      • Davies D.R.
      Crystal structures of a mutant (βK87T) tryptophan synthase α2β2 complex with ligands bound to the active sites of the α- and β-subunits reveal ligand-induced conformational changes.
      ). We characterized several mutants in collaboration with Michael Dunn, including the β subunit E109D mutant (
      • Brzović P.S.
      • Kayastha A.M.
      • Miles E.W.
      • Dunn M.F.
      Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the β-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium.
      ). This mutant also has been used in studies of channeling (see below). We are especially interested in the functional role of a flexible loop (loop 6) in the α subunit because loop 6 contains the site of limited tryptic cleavage at Arg-188 that we discovered earlier (
      • Higgins W.
      • Fairwell T.
      • Miles E.W.
      An active proteolytic derivative of the α subunit of tryptophan synthase. Identification of the site of cleavage and characterization of the fragments.
      ). Studies with the α subunit R179L mutant showed that loop 6 is important both for ligand binding to the α site and for the ligand-induced conformational change from an “open” to a “closed” structure (
      • Brzović P.S.
      • Sawa Y.
      • Hyde C.C.
      • Miles E.W.
      • Dunn M.F.
      Evidence that mutations in a loop region of the α-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex.
      ). Our results showed that Thr-183 in α subunit loop 6 also plays a role in allosteric regulation (
      • Yang X.J.
      • Miles E.W.
      Threonine 183 and adjacent flexible loop residues in the tryptophan synthase α subunit have critical roles in modulating the enzymatic activities of the β subunit in the α2β2 complex.
      ).

      Channeling and Intersubunit Communication

      The tryptophan synthase α2β2 complex is thought to channel indole, which is an intermediate in the αβ reaction (Equation 3), from the active site of the α subunit to the active site of the β subunit. The three-dimensional structure of the tryptophan synthase α2β2 complex (
      • Hyde C.C.
      • Ahmed S.A.
      • Padlan E.A.
      • Miles E.W.
      • Davies D.R.
      Three-dimensional structure of the tryptophan synthase α2β2 multienzyme complex from Salmonella typhimurium.
      ) provides physical evidence for a 25-Å hydrophobic tunnel that connects the α and β subunits (Fig. 5). We probed the role of the tunnel in indole channeling and in intersubunit communication by kinetic characterization of wild-type and mutant forms of the tryptophan synthase α2β2 complex (
      • Anderson K.S.
      • Miles E.W.
      • Johnson K.A.
      Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism.
      ). Rapid chemical quench flow experiments showed that indole is channeled rapidly and that l-serine at the β site increases the rate of indole-3-glycerol phosphate cleavage at the α site. A mutation at the β site (E109D) slows the reaction rate enough to permit detection of the indole intermediate in a single turnover in the αβ reaction. Rapid kinetic analyses of the α2β2 complex with mutations in the β site (C170F and C170W) designed to restrict the tunnel showed that the mutations interfere with efficient indole channeling such that indole can be detected in a single turnover (
      • Anderson K.S.
      • Kim A.Y.
      • Quillen J.M.
      • Sayers E.
      • Yang X.J.
      • Miles E.W.
      Kinetic characterization of channel impaired mutants of tryptophan synthase.
      ). We also probed the indole tunnel by Nile Red fluorescence with wild-type, mutant, and chemically modified enzymes (Fig. 6) (
      • Ruvinov S.B.
      • Yang X.J.
      • Parris K.D.
      • Banik U.
      • Ahmed S.A.
      • Miles E.W.
      • Sackett D.L.
      Ligand-mediated changes in the tryptophan synthase indole tunnel probed by Nile Red fluorescence with wild type, mutant, and chemically modified enzymes.
      ). The interaction of Nile Red in the nonpolar tunnel near Cys-170 and Phe-280 in the β site is supported by experiments with residues altered at these positions by mutation or chemical modification. The results of our experiments with Nile Red as a probe of conformational changes in the tunnel suggest that allosteric ligands and active site ligands induce a tunnel restriction near Phe-280 that controls the passage of indole. Thus, the rapid kinetic experiments and the experiments with Nile Red provide evidence for ligand-dependent intersubunit communication.
      Figure thumbnail gr6
      FIGURE 6Edith Miles and her associates enjoy lunch at a Thai restaurant in 1997. Left to right, Li-hong Yang, Roger Rowlett, Peter McPhie, Kwang-Hwan Jhee, and Edith.

      Allosteric Regulation

      We and others have found that pH, temperature, cations, and α subunit ligands regulate the tryptophan synthase α2β2 complex by altering the equilibrium distribution of PLP intermediates with serine (
      • Peracchi A.
      • Mozzarelli A.
      • Rossi G.L.
      Monovalent citations affect dynamic and functional properties of the tryptophan synthase α2β2 complex.
      ,
      • Peracchi A.
      • Bettati S.
      • Mozzarelli A.
      • Rossi G.L.
      • Miles E.W.
      • Dunn M.F.
      Allosteric regulation of tryptophan synthase: effects of pH, temperature, and α-subunit ligands on the equilibrium distribution of pyridoxal 5′-phosphate-l-serine intermediates.
      ,
      • Fan Y.X.
      • McPhie P.
      • Miles E.W.
      Regulation of tryptophan synthase by temperature, monovalent cations, and an allosteric ligand. Evidence from Arrhenius plots, absorption spectra, and primary kinetic isotope effects.
      ). The results support a model in which reaction conditions alter the equilibrium distribution between a low-activity open conformation and a high-activity closed conformation. The aminoacrylate PLP Schiff base is associated with the closed form and is stabilized by temperature, protons, and α subunit ligands.

      Status of Tryptophan Synthase Research Today

      I am gratified that investigations of tryptophan synthase continue to bring rich rewards. A recent review (
      • Dunn M.F.
      Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex.
      ) summarizes the current status of research and presents evidence for the allosteric regulation of substrate channeling and catalysis in the tryptophan synthase α2β2 complex. The catalytic pathway is composed of a series of at least nine reactions involving PLP and l-serine at the β site and reactions with the indole ring. Advances in understanding the relationship between chemical events at the α and β sites and allosteric regulation have been greatly aided by the synthesis of substrate analogs and characterization of the interactions of these analogs with the α and β sites by x-ray crystallography and kinetic analyses (
      • Dunn M.F.
      Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex.
      ). The most interesting of these structures involves the reaction of indoline at the α site with bound glyceraldehyde 3-phosphate. The combination of indole-3-glycerol phosphate binding to the α site and the reaction of l-serine to form aminoacrylate at the β site gives a crystal structure with both α and β sites closed. Dunn (
      • Dunn M.F.
      Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex.
      ) concluded that ligand binding at the α and β sites and allosteric interactions acting over 25 Å choreograph the switching of the α and β subunits between low-activity open conformations and high-activity closed conformations. The conformational switches synchronize the activities of the α and β subunits and lead to efficient channeling of indole and overall catalysis.

      Mentors, Models, and Important Professional Contacts

      Herbert Tabor has been my most important mentor at NIH (Fig. 7). He was chief of the Laboratory of Biochemical Pharmacology when I joined the laboratory in 1966. He encouraged me to set my own course in independent research. Herb has always treated men and women equally and fairly. Several women in his laboratory have inspired me: Celia Tabor, Loretta Leive, Nancy Nossal, and Claude Klee. Other distinguished women scientists at NIH also have served as role models: Thressa Stadtman, Maxine Singer, Elizabeth Neufeld, Ruth Kirschstein, and Ann Ginsburg. I have enjoyed fruitful collaborations with several NIH scientists, including Robert Phillips, Peter McPhie, Allen Minton, David Davies, Craig Hyde, Ashraf Ahmed, Sangkee Rhee, and Boon Chock. Mentors and co-workers outside of NIH include Charles Yanofsky, Irving Crawford, Ronald Bauerle, Kasper Kirschner, Michael Goldberg, Michael Dunn, Karen Anderson, Kenneth Johnson, Katsuhide Yutani, Andrea Mozzarelli, and Ilme Schlichting.
      Figure thumbnail gr7
      FIGURE 7Edith Miles presents the Association for Women in Science Bethesda Chapter Annual Award for Excellence in Mentoring to Celia Tabor and Herbert Tabor at a meeting in 1998.
      I developed many professional contacts at meetings, including Charles Yanofsky's tryptophan meetings at Stanford University and Asilomar and B6 meetings in Moscow, Leningrad, Finland, Japan, and Capri. I have visited Japan several times in connection with a research grant with Katsuhide Yutani and several meetings. I have had contacts with many Japanese scientists, including Kenji Soda, Hideaki Yamada, Hidehiko Kumagai, Hiroshi Wada, Osao Adachi, and Katsuyuki Tanizawa.

      Family and Activities

      I met my husband, Harry Todd Miles, at a Federation of American Societies for Experimental Biology (FASEB) meeting in Atlantic City in 1964. Todd was at the meeting with his research associate Frank Howard, whom I had known when we were both graduate students at UC-Berkeley. Todd was chief of the Organic Chemistry Section in the Laboratory of Molecular Biology at NIDDK. We married in 1966 and had two sons in 1969 and 1971. I thank Todd for being an excellent co-parent and for encouraging me to continue in research. We were fortunate to be able to enter our sons into the NIH preschool in 1974 a few months after it opened. Todd and I were both involved in the Parents of Preschoolers, Inc. (POPI), when it established parental governance of the preschool in 1975. Our sons attended Ayrlawn Elementary School and continued in the POPI afterschool program at Ayrlawn. All four of us drove together to our work and school in the morning and home together in the evening for seven years. Our sons are now well educated and have rewarding careers and families. Todd and I both retired on September 30, 2000, and are Scientists Emeritus. We enjoy classes and travel together and visits with our children and grandchildren. I am doing hand-building pottery at Glen Echo. I am active with the Bethesda Chapter of the Association for Women in Science as a mentor and program organizer. I work with my neighborhood association to help seniors continue to live in their homes (Aging in Place).

      Acknowledgments

      I thank my postdoctoral fellows, collaborators, friends, mentors, and family for inspiration and encouragement. I especially thank Herbert Tabor and NIH for providing research facilities and a good working environment. The noontime seminars and journal clubs in our laboratory had a strong influence on all of us.

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