Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium

The three-dimensional structure of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium has been determined by x-ray crystallography at 2.5 A resolution. The four polypeptide chains are arranged nearly linearly in an alpha beta beta alpha order forming a complex 150 A long. The overall polypeptide fold of the smaller alpha subunit, which cleaves indole glycerol phosphate, is that of an 8-fold alpha/beta barrel. The alpha subunit active site has been located by difference Fourier analysis of the binding of indole propanol phosphate, a competitive inhibitor of the alpha subunit and a close structural analog of the natural substrate. The larger pyridoxal phosphate-dependent beta subunit contains two domains of nearly equal size, folded into similar helix/sheet/helix structures. The binding site for the coenzyme pyridoxal phosphate lies deep within the interface between the two beta subunit domains. The active sites of neighboring alpha and beta subunits are separated by a distance of about 25 A. A tunnel with a diameter matching that of the intermediate substrate indole connects these active sites. The tunnel is believed to facilitate the diffusion of indole from its point of production in the alpha subunit active site to the site of tryptophan synthesis in the beta active site and thereby prevent its escape to the solvent during catalysis.     

Conformational changes in the alpha-subunit coupled to binding of the beta 2-subunit of tryptophan synthase from Escherichia coli: crystal structure of the tryptophan synthase alpha-subunit alone

When the tryptophan synthase alpha- and beta(2)-subunits combine to form the alpha(2)beta(2)-complex, the enzymatic activity of each subunit is stimulated by 1-2 orders of magnitude. To elucidate the structural basis of this mutual activation, it is necessary to determine the structures of the alpha- and beta-subunits alone and together with the alpha(2)beta(2)-complex. The crystal structures of the tryptophan synthase alpha(2)beta(2)-complex from Salmonella typhimurium (Stalpha(2)beta(2)-complex) have already been reported. However, the structures of the subunit alone from mesophiles have not yet been determined. The structure of the tryptophan synthase alpha-subunit alone from Escherichia coli (Ecalpha-subunit) was determined by an X-ray crystallographic analysis at 2.3 A, which is the first report on the subunits alone from the mesophiles. The biggest difference between the structures of the Ecalpha-subunit alone and the alpha-subunit in the Stalpha(2)beta(2)-complex (Stalpha-subunit) was as follows. Helix 2' in the Stalpha-subunit, including an active site residue (Asp60), was changed to a flexible loop in the Ecalpha-subunit alone. The conversion of the helix to a loop resulted in the collapse of the correct active site conformation. This region is also an important part for the mutual activation in the Stalpha(2)beta(2)-complex and interaction with the beta-subunit. These results suggest that the formation of helix 2'that is essential for the stimulation of the enzymatic activity of the alpha-subunit is constructed by the induced-fit mode involved in conformational changes upon interaction between the alpha- and beta-subunits. This also confirms the prediction of the conformational changes based on the thermodynamic analysis for the association between the alpha- and beta-subunits.     

The crystal structure of the tryptophan synthase beta subunit from the hyperthermophile Pyrococcus furiosus. Investigation of stabilization factors.

The structure of the tryptophan synthase beta2 subunit (Pfbeta2) from the hyperthermophile, Pyrococcus furiosus, was determined by X-ray crystallographic analysis at 2.2 A resolution, and its stability was examined by DSC. This is the first report of the X-ray structure of the tryptophan synthase beta2 subunit alone, although the structure of the tryptophan synthase alpha2beta2 complex from Salmonella typhimurium has already been reported. The structure of Pfbeta2 was essentially similar to that of the beta2 subunit (Stbeta2) in the alpha2beta2 complex from S. typhimurium. The sequence alignment with secondary structures of Pfbeta and Stbeta in monomeric form showed that six residues in the N-terminal region and three residues in the C-terminal region were deleted in Pfbeta, and one residue at Pro366 of Stbeta and at Ile63 of Pfbeta was inserted. The denaturation temperature of Pfbeta2 was higher by 35 degrees C than the reported values from mesophiles at approximately pH 8. On the basis of structural information on both proteins, the analyses of the contributions of each stabilization factor indicate that: (a) the higher stability of Pfbeta2 is not caused by either a hydrophobic interaction or an increase in ion pairs; (b) the number of hydrogen bonds involved in the main chains of Pfbeta is greater by about 10% than that of Stbeta, indicating that the secondary structures of Pfbeta are more stabilized than those of Stbeta and (c) the sequence of Pfbeta seems to be better fitted to an ideally stable structure than that of Stbeta, as assessed from X-ray structure data.     

Confinement and crowding effects on tryptophan synthase alpha2beta2 complex

Biological molecules experience in vivo a highly crowded environment. The investigation of the functional properties of the tryptophan synthase alpha(2)beta(2) complex either entrapped in wet nanoporous silica gels or in the presence of the crowding agents dextran 70 and ficoll 70 indicates that the rates of the conformational transitions associated to catalysis and regulation are reduced, and an open and less catalytically active conformation is stabilized.     

Microcrystals of tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium are catalytically active

An improved and efficient method has been developed for the purification of the tryptophan synthase alpha 2 beta 2 complex (EC 4.2.1.20) from Salmonella typhimurium containing a multicopy plasmid. Microcrystals prepared in 12% poly(ethylene glycol) 8000 containing 2.5 mM spermine are shown by scanning electron microscopy to have the same crystal habit as the larger crystals that are being used for structural analysis by X-ray crystallography. The average dimensions of the crystals are 33 microns (length) X 9 microns (width) X 3 microns (maximum thickness). Our finding that suspensions of microcrystals are active in several reactions catalyzed by the active sites of the alpha and beta 2 subunits demonstrates that both active sites are functional in the crystal and accessible to substrates. Thus the larger crystals being used for X-ray crystallographic studies should form complexes with substrates and analogues at both active sites and should yield functionally relevant structural information. A comparison of the reaction rates of suspensions of microcrystals with those of the soluble enzyme shows that the maximum rate of the crystalline enzyme is 0.8 that of the soluble enzyme in the cleavage of indole-3-glycerol phosphate (alpha reaction), 0.3 that of the soluble enzyme in the synthesis of L-tryptophan by the beta reaction or the coupled alpha beta reaction, and 2.7 that of the soluble enzyme in the serine deaminase reaction. These small differences in rates probably reflect functional differences between the crystalline and soluble enzymes since the reaction rates of the microcrystals are calculated to be virtually free of diffusional limitation under these reaction conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microspectrophotometric studies on single crystals of the tryptophan synthase alpha 2 beta 2 complex demonstrate formation of enzyme-substrate intermediates

Microspectrophotometry of single crystals of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium is used to compare the catalytic and regulatory properties of the enzyme in the soluble and crystalline states. Polarized absorption spectra demonstrate that chromophoric intermediates are formed between pyridoxal phosphate at the active site of the beta subunit and added substrates, substrate analogs, and reaction intermediate analogs. Although the crystalline and soluble forms of the enzyme produce some of the same enzyme-substrate intermediates, including Schiff base and quinonoid intermediates, in some cases the equilibrium distribution of these intermediates differs in the two states of the enzyme. Ligands which bind to the active site of the alpha subunit alter the distribution of intermediates formed at the active site of the beta subunit in both the crystalline and soluble states. The three-dimensional structures of the tryptophan synthase alpha 2 beta 2 complex and of a derivative with indole-3-propanol phosphate bound at the active site of the alpha subunit have recently been reported (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 264, 17857-17871). Our present findings help to establish experimental conditions for selecting defined intermediates for future x-ray crystallographic analysis of the alpha 2 beta 2 complex with ligands bound at the active sites of both alpha and beta subunits. These crystallographic studies should explain how catalysis occurs at the active site of the beta subunit and how the binding of a ligand to one active site affects the binding of a ligand to the other active site which is 25 A away.     

5-Azidoindole binding to the Escherichia coli tryptophan synthase alpha 2 beta 2 complex

The tryptophan synthase alpha 2 beta 2 complex catalyzes tryptophan (Trp) biosynthesis from serine plus either indole (IN) or indole-3-glycerol phosphate (InGP). The photoreactive 5-azido analog in IN (AzIN), itself a substrate in the dark, was utilized to examine the substrate binding sites on this enzyme. When irradiated with AzIN at concentrations approaching IN saturation for the IN----Trp activity (0.1 mM), in the absence of serine, the enzyme was increasingly inactivated (up to 70-80%) concomitant with the progressive binding of a net of 2 mol AzIN per alpha beta equivalent. Little or no cooperativity in the binding of the 2 mol AzIN was observed. In contrast, there was minimal effect on the IN----InGP activity. Under these conditions AzIN appeared to be incorporated equally into each subunit. No significant inactivation nor binding occurred in the presence of serine. A quantitatively similar inactivation of InGP----Trp activity was observed over the same AzIN concentration range, suggesting common IN sites for Trp biosynthesis from either indole substrate. At higher concentrations (0.1-0.7 mM), no further inactivation occurred, although there was extensive additional binding (up to 10 mol/alpha beta equivalent). These data are consistent, although more clear-cut quantitatively, with the high- and low-affinity sites proposed from equilibrium dialysis studies. AzIN binding studies utilizing the isolated beta 2 subunit confirmed earlier reports suggesting the existence of many nonspecific IN binding sites on this subunit.     

Crystallization and preliminary X-ray crystallographic data of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium

The tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium can be crystallized by the method of vapor diffusion from solutions of polyethylene glycol 8000 and various salts. Thin needles are obtained in the presence of a monovalent cation (Na+), thicker crystals are obtained in the presence of divalent cations (Mg2+, Mn2+, Fe2+, Ca2+, Zn2+), and square-faced crystals are obtained in the presence of spermine. Although the spermine and Mg2+ crystals differ in morphology, they are both monoclinic and in space group C2 with a = 184.5 A, b = 62.4 A, c = 67.7 A, beta = 94 degrees 40', and one alpha beta pair of Mr = 71,700/asymmetric unit. The crystals appear reasonably resistant to radiation damage and should be suitable for a complete structure investigation. The separated S. typhimurium alpha, holo-beta 2, and apo-beta 2 subunits do not crystallize under these conditions nor do the alpha 2 beta 2 complex or the alpha- or holo-beta 2 subunits from Escherichia coli or from an interspecies hybrid.     

The tryptophan synthase alpha 2 beta 2 complex. Cleavage of a flexible loop in the alpha subunit alters allosteric properties

This study explores the catalytic and allosteric roles of a flexible loop in tryptophan synthase. Trypsin is known to cleave the tryptophan synthase alpha 2 beta 2 complex in an alpha subunit loop at Arg-188. Cleavage yields an active "nicked" alpha 2 beta 2 derivative. The new results provide evidence that the alpha subunit loop serves two important roles: substrate binding and communicating the effects of substrate binding to the beta subunit. A role for the loop in substrate binding is supported by our finding that addition of a substrate analogue of the alpha subunit, alpha-glycerol 3-phosphate, decreases the rate of cleavage by trypsin. An allosteric role for the loop is supported by the finding although the native alpha 2 beta 2 complex is strongly inhibited by alpha-glycerol 3-phosphate, the nicked alpha 2 beta 2 complex is desensitized to this inhibition. The time course of proteolysis in the presence and absence of alpha-glycerol 3-phosphate is followed by sodium dodecyl sulfate-gel electrophoresis and by assays of activity in the presence and absence of alpha-glycerol 3-phosphate. We use spectroscopic measurements of the pyridoxal phosphate-L-tryptophan intermediates at the active site of the beta subunit to determine the affinity of the native and nicked enzymes for L-tryptophan and alpha-glycerol 3-phosphate. Although cleavage alters the equilibrium distribution of intermediates and reduces the affinity for alpha-glycerol 3-phosphate, it has little effect on the affinity for amino acids bound to the beta subunit. We conclude that the loop in the alpha subunit is important for ligand binding and for communicating the effects of ligand binding from the alpha subunit to the beta subunit in the alpha 2 beta 2 complex.     

The tryptophan synthase alpha 2 beta 2 complex: a comparison of the reactivity of amino groups in the alpha and beta 2 subunits and in the complex by differential labeling studies

The interaction of the alpha and beta 2 subunits of tryptophan synthase of Escherichia coli to form an alpha 2 beta 2 complex has been probed by differential labeling studies. In the first step the separate alpha or beta 2 subunit or the alpha 2 beta 2 complex was labeled by reductive methylation with trace amounts of [3H]HCHO in the presence of NaCNBH3. In the second step the 3H-labeled preparation was fully labeled under denaturing conditions with [14C]HCHO and NaCNBH3. Peptides containing labeled monomethyl or dimethyl amino groups were isolated after thermolytic digestion or after cyanogen bromide treatment. The 3H/14C ratio of each peptide is a measure of the relative reactivity of the amino group or groups in each peptide. The most reactive amino group in the alpha subunit, lysine-109, is strongly shielded from modification in the alpha 2 beta 2 complex. The most reactive amino group in the beta 2 subunit, the amino-terminal threonine, is not shielded from modification in the alpha 2 beta 2 complex.     

Effect of pH and monovalent cations on the formation of quinonoid intermediates of the tryptophan synthase alpha(2)beta(2) complex in solution and in the crystal

Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate-dependent enzymes. Whereas the structures of other pyridoxal 5'-phosphate-bound intermediates have been determined, the structure of a quinonoid species has not yet been reported. Here, we investigate factors controlling the accumulation and stability of quinonoids formed at the beta-active site of tryptophan synthase both in solution and the crystal. The quinonoids were obtained by reacting the alpha-aminoacrylate Schiff base with different nucleophiles, focusing mainly on the substrate analogs indoline and beta-mercaptoethanol. In solution, both monovalent cations (Cs(+) or Na(+)) and alkaline pH increase the apparent affinity of indoline and favor accumulation of the indoline quinonoid. A similar pH dependence is observed when beta-mercaptoethanol is used. As indoline and beta-mercaptoethanol exhibit very distinct ionization properties, this finding suggests that nucleophile binding and quinonoid stability are controlled by some ionizable protein residue(s). In the crystal, alkaline pH favors formation of the indoline quinonoid as in solution, but the effect of cations is markedly different. In the absence of monovalent metal ions the quinonoid species accumulates substantially, whereas in the presence of sodium ions the accumulation is modest, unless alpha-subunit ligands are also present. Alpha-subunit ligands not only favor the formation of the intermediate, but also reduce significantly its decay rate. These findings define experimental conditions suitable for the stabilization of the quinonoid species in the crystal, a critical prerequisite for the determination of the three-dimensional structure of this intermediate.     

Photoinactivation and photoaffinity labeling of tryptophan synthase alpha 2 beta 2 complex by the product analogue 6-azido-L-tryptophan

The photoaffinity reagent 6-azido-L-tryptophan was synthesized by chemical methods. It binds reversibly in the dark to the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli and forms a quinonoid intermediate with enzyme-bound pyridoxal phosphate (lambda max = 476 nm). The absorbance of this chromophore has been used for spectrophotometric titrations to determine the binding of 6-azido-L-tryptophan (the half-saturation value [S]0.5 = 6.3 microM). Photolysis of the quinonoid form of the alpha 2 beta 2 complex results in time-dependent inactivation of the beta 2 subunit but not of the alpha subunit. The extent of photoinactivation is directly proportional to the absorbance at 476 nm of the quinonoid intermediate prior to photolysis. The substrate L-serine is a competitive inhibitor of 6-azido-L-tryptophan binding and photoinactivation. The competitive inhibitors L-tryptophan, D-tryptophan, and oxindolyl-L-alanine also protect against photoinactivation. The results demonstrate that 6-azido-L-tryptophan is a quasi-substrate for the alpha 2 beta 2 complex of tryptophan synthase and that photolysis of the enzyme-quasi-substrate quinonoid intermediate results in photoinactivation. The modified alpha 2 beta 2 complex retains its ability to bind pyridoxal phosphate and to cleave indole-3-glycerol phosphate, a reaction catalyzed by the alpha subunit. 6-Azido-L-tryptophan (side-chain 1,2,3-14C3 labeled) was synthesized enzymatically from 6-azidoindole and uniformly labeled L-[14C]serine by the alpha 2 beta 2 complex of tryptophan synthase on a preparative scale and has been isolated. Incorporation of 14C label from 6-azido-L-[14C]tryptophan is stoichiometric with inactivation. Our finding that most of the incorporated 14C label is bound in an unstable linkage suggests that an active site carboxyl residue is the major site of photoaffinity labeling by 6-azido-L-tryptophan.     

Stereochemistry and mechanism of a new single-turnover, half-transamination reaction catalyzed by the tryptophan synthase alpha 2 beta 2 complex

Tryptophan synthase is a versatile enzyme that catalyzes a wide variety of pyridoxal phosphate dependent reactions that are also catalyzed in model systems. These include beta-replacement, beta-elimination, racemization, and transamination reactions. We now show that the apo-alpha 2 beta 2 complex of tryptophan synthase will bind two unnatural substrates, pyridoxamine phosphate and indole-3-pyruvic acid, and will convert them by a single-turnover, half-transamination reaction to pyridoxal phosphate and L-tryptophan, the natural coenzyme and a natural product, respectively. This enzyme-catalyzed reaction is more rapid and more stereospecific than an analogous model reaction. The pro-S 4'-methylene proton of pyridoxamine phosphate is removed during the reaction, and the product is primarily L-tryptophan. We conclude that pyridoxal phosphate enzymes may be able to catalyze some unnatural reactions involving bound reactants and bound coenzyme since the coenzyme itself has the intrinsic ability to promote a variety of reactions.     

Effects of modification of the beta 2 subunit and of the alpha2beta2 complex of tryptophan synthase by alpha-cyanoglycine, a substrate analog.

α-Cyanoglycine inactivates the pyridoxal-P forms of the β₂ subunit and the α₂β₂ complex of tryptophan synthase; an intense chromophore at 430 nm forms concomitantly. The slow reactivation of the modified β₂ subunit upon dialysis ($\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 24 hours}$) is prevented by addition of α subunit, which presumably acts by changing the environment of the chromophoric derivative. These data and the observed protection from inhibition by L-serine indicate that α-cyanoglycine acts as a substrate analog which undergoes a second, largely irreversible reaction at the active site of the β₂ subunit. Modification of the β₂ subunit increases its affinity for the α subunit. Modification of the α₂β₂ complex increases its stability to heat, urea, and low pH.     

Structure and function of the tryptophan synthase alpha(2)beta(2) complex. Roles of beta subunit histidine 86

To probe the structural and functional roles of active-site residues in the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium, we have determined the effects of mutation of His(86) in the beta subunit. His(86) is located adjacent to beta subunit Lys(87), which forms an internal aldimine with the pyridoxal phosphate and catalyzes the abstraction of the alpha-proton of L-serine. The replacement of His(86) by leucine (H86L) weakened pyridoxal phosphate binding approximately 20-fold and abolished the circular dichroism signals of the bound coenzyme and of a reaction intermediate. Correlation of these results with previous crystal structures indicates that beta-His(86) plays a structural role in binding pyridoxal phosphate and in stabilizing the correct orientation of pyridoxal phosphate in the active site of the beta subunit. The H86L mutation also altered the pH profiles of absorbance and fluorescence signals and shifted the pH optimum for the synthesis of L-tryptophan from pH 7.5 to 8.8. We propose that the interaction of His(86) with the phosphate of pyridoxal phosphate and with Lys(87) lowers the pK(a) of Lys(87) in the wild-type alpha(2)beta(2) complex and thereby facilitates catalysis by Lys(87) in the physiological pH range.     

Novel allosteric effectors of the tryptophan synthase alpha(2)beta(2) complex identified by computer-assisted molecular modeling

Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the formation of L-tryptophan. The functional properties of one subunit are allosterically regulated by ligands of the other subunit. Molecules tailored for binding to the alpha-active site were designed using as a starting model the three-dimensional structure of the complex between the enzyme from Salmonella typhimurium and the substrate analog indole-3-propanol phosphate. On the basis of molecular dynamics simulations, indole-3-acetyl-X, where X is glycine, alanine, valine and aspartate, and a few other structurally related compounds were found to be good candidates for ligands of the alpha-subunit. The binding of the designed compounds to the alpha-active site was evaluated by measuring the inhibition of the alpha-reaction of the enzyme from Salmonella typhimurium. The inhibition constants were found to vary between 0.3 and 1.7 mM. These alpha-subunit ligands do not bind to the beta-subunit, as indicated by the absence of effects on the rate of the beta-reaction in the isolated beta(2) dimer. A small inhibitory effect on the activity of the alpha(2)beta(2) complex was caused by indole-3-acetyl-glycine and indole-3-acetyl-aspartate whereas a small stimulatory effect was caused by indole-3-acetamide. Furthermore, indole-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide perturb the equilibrium of the catalytic intermediates formed at the beta-active site, stabilizing the alpha-aminoacrylate Schiff base. These results indicate that (i) indole-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide bind to the alpha-subunit and act as allosteric effectors whereas indole-3-acetyl-valine and indole-3-acetyl-alanine only bind to the alpha-subunit, and (ii) the terminal phosphate present in the already known allosteric effectors of tryptophan synthase is not strictly required for the transmission of regulatory signals.     

Origin of the mutual activation of the alpha and beta 2 subunits in the alpha 2 beta 2 complex of tryptophan synthase. Effect of alanine or glycine substitutions at proline residues in the alpha subunit.

To understand how the alpha and beta 2 subunits of tryptophan synthase from Escherichia coli interact to form an alpha 2 beta 2 complex and undergo mutual activation, we have investigated alpha subunits with single amino acid replacements at conserved proline residues. Although the activities of alpha 2 beta 2 complexes that contain wild type alpha subunit or alpha subunits substituted at positions 28, 62, 96, and 207 are similar, the activities of alpha 2 beta 2 complexes that contain alpha subunits substituted at positions 57 and 132 are remarkably altered. Whereas the latter enzymes have greatly reduced activities in the individual half-reactions, they have considerably higher activities in the overall reaction. These remarkable activity results are explained by a decrease in the affinity of these mutant alpha subunits for the beta 2 subunit and by an increase in the affinity in the combined presence of ligands of both the alpha subunit and the beta 2 subunit. Isothermal calorimetric titrations of wild type beta 2 subunit with wild type alpha subunit and a mutant alpha subunit containing a substitution of glycine for proline at position 132 show that both the affinity and the exothermic association enthalpy are greatly reduced in the mutant alpha subunit although the stoichiometry of association is unchanged. The affinity of the mutant alpha subunit for the beta 2 subunits is greatly increased in the presence of an alpha subunit ligand, alpha-glycerol phosphate. We conclude that proline 132 plays a critical role in subunit interaction and in mutual subunit activation.     

Threonine 183 and adjacent flexible loop residues in the tryptophan synthase alpha subunit have critical roles in modulating the enzymatic activities of the beta subunit in the alpha 2 beta 2 complex.

This study investigates the catalytic and allosteric roles of a flexible loop in the tryptophan synthase alpha 2 beta 2 complex. This loop connects helix 6 and strand 6 in the alpha subunit, an 8-fold alpha/beta barrel polypeptide. We have engineered three mutations in this disordered loop: a deletion of residues 185-187 and the replacement of threonine 183 by serine (T183S) or by alanine (T183A). Position 183 is a site of an inactivating mutation identified by Yanofsky's group (Yanofsky, C., Drapeau, G. R., Guest, J. R., and Carlton, B. C. (1967) Proc. Natl. Acad. Sci. U.S.A. 57, 296-298). The three engineered alpha subunits form stable, stoichiometric alpha 2 beta 2 complexes with the beta subunit which bind alpha and beta subunit ligands. Although changing threonine 183 to serine has little effect on the enzymatic properties, changing threonine 183 to alanine or deleting residues 185-187 results in a 50-fold reduction in the intrinsic activity of the alpha subunit alone and in the alpha site activity of the alpha 2 beta 2 complex. The latter two mutations profoundly alter the way in which the alpha subunit modulates the spectral properties and the activities of the wild-type beta subunit. These mutations also eliminate the effects of alpha subunit ligands on the beta subunit. Although the beta subunit ligand, L-serine, greatly stabilizes the wild-type alpha 2 beta 2 complex to dissociation and to proteolysis, L-serine stabilizes the T183A alpha 2 beta 2 complex weakly or not at all. Our findings suggest that the hydroxyl residue at position 183 and the adjacent residues in the alpha subunit loop play critical roles in the reciprocal communication between the alpha and beta subunits in the alpha 2 beta 2 complex. The results also help to explain how the wild-type alpha subunit or ammonium ion modulates the activities of the beta subunit.     

Prostaglandin F2alpha formation from prostaglandin H2 by prostaglandin F synthase (PGFS): crystal structure of PGFS containing bimatoprost

Prostaglandin H(2) (PGH(2)) formed from arachidonic acid is an unstable intermediate and is efficiently converted into more stable arachidonate metabolites by the action of enzymes. Prostaglandin F synthase (PGFS) has dual catalytic activities: formation of PGF(2)(alpha) from PGH(2) by the PGH(2) 9,11-endoperoxide reductase activity and 9alpha,11beta-PGF(2) (PGF(2)(alphabeta)) from PGD(2) by the PGD(2) 11-ketoreductase activity in the presence of NADPH. Bimatoprost (BMP), which is a highly effective ocular hypotensive agent, is a PGF(2)(alpha) analogue that inhibits both the PGD(2) 11-ketoreductase and PGH(2) 9,11-endoperoxide reductase activities of PGFS. To examine the catalytic mechanism of PGH(2) 9,11-endoperoxide reductase, a crystal structure of PGFS[NADPH + BMP] has been determined at 2.0 A resolution. BMP binds near the PGD(2) binding site, but the alpha- and omega-chains of BMP are locate on the omega- and alpha-chains of PGD(2), respectively. Consequently, the bound BMP and PGD(2) direct their opposite faces of the cyclopentane moieties toward the nicotinamide ring of the bound NADP. The alpha- and omega-chains of BMP are involved in H-bonding with protein residues, while the cyclopentane moiety is surrounded by water molecules and is not directly attached to either the protein or the bound NADPH, indicating that the cyclopentane moiety is movable in the active site. From the complex structure, two model structures of PGFS containing PGF(2)(alpha) and PGH(2) were built. On the basis of the model structures and inhibition data, a putative catalytic mechanism of PGH(2) 9,11-endoperoxide reductase of PGFS is proposed. Formation of PGF(2)(alpha) from PGH(2) most likely involves a direct hydride transfer from the bound NADPH to the endoperoxide of PGH(2) without the participation of specific amino acid residues.