Search

Purpose: The purpose of this study was to characterize the 2-deoxy-2-[18F]fluoro-D-glucose (FDG) PET/CT imaging findings of sacral metastatic meningioma., Materials and Methods: An 18-year-old woman with a history of metastatic meningioma, who presented with intractable pain and spasm of the right lower extremity, underwent FDG PET/CT imaging., Results: FDG PET/CT scan demonstrated a moderately hypermetabolic sacral mass. The biopsy of the lesion was consistent with a high-grade malignant meningioma., Conclusions: Although meningioma is typically a benign tumor, in rare instances, it metastasizes. The current case example exhibits moderately increased metabolism despite findings of high-grade malignancy on biopsy.

Transglutaminases(TGases; protein-glutamine-glutamyl-transferases) are a large family of calcium-dependent acyl-transfer enzymes that catalyze the formation of covalent cross links in proteins. Of these, the "epidermal" or "hair follicle" TGase 3 isoform is critically involved in barrier formation in epithelia. It is a zymogen, requiring proteolytic activation to achieve maximal specific activity. In order to understand its structure and function, we have devised methods for the rapid large-scale expression of the TGase 3 zymogen in the baculovirus system, and here we describe the purification of the zymogen and activated forms. We describe methods for the formation of high-quality, well-diffracting crystals within 3-5 days, using both dioxane and beta-octylglucoside to overcome severe twinning problems. The crystal of the zymogen belongs to the triclinic space group P1 and diffracts to 2.2-A resolution, and the crystal of the active form belongs to the P2(1) space group at 2.7-A resolution.

The bacteriophage T4 gene 59 helicase assembly protein is required for recombination-dependent DNA replication, which is the predominant mode of DNA replication in the late stage of T4 infection. T4 gene 59 helicase assembly protein accelerates the loading of the T4 gene 41 helicase during DNA synthesis by the T4 replication system in vitro. T4 gene 59 helicase assembly protein binds to both T4 gene 41 helicase and T4 gene 32 single-stranded DNA binding protein, and to single and double-stranded DNA. We show here that T4 gene 59 helicase assembly protein binds most tightly to fork DNA substrates, with either single or almost entirely double-stranded arms. Our studies suggest that the helicase assembly protein is responsible for loading T4 gene 41 helicase specifically at replication forks, and that its binding sites for each arm must hold more than six, but not more than 12 nucleotides. The 1.45 A resolution crystal structure of the full-length 217-residue monomeric T4 gene 59 helicase assembly protein reveals a novel alpha-helical bundle fold with two domains of similar size. Surface residues are predominantly basic (pI 9.37) with clusters of acidic residues but exposed hydrophobic residues suggest sites for potential contact with DNA and with other protein molecules. The N-terminal domain has structural similarity to the double-stranded DNA binding domain of rat HMG1A. We propose a speculative model of how the T4 gene 59 helicase assembly protein might bind to fork DNA based on the similarity to HMG1, the location of the basic and hydrophobic regions, and the site size of the fork arms needed for tight fork DNA binding. The fork-binding model suggests putative binding sites for the T4 gene 32 single-stranded DNA binding protein and for the hexameric T4 gene 41 helicase assembly.

Integration of retroviral cDNA is a necessary step in viral replication. The virally encoded integrase protein and DNA sequences at the ends of the linear viral cDNA are required for this reaction. Previous studies revealed that truncated forms of Rous sarcoma virus integrase containing two of the three protein domains can carry out integration reactions in vitro. Here, we describe the crystal structure at 2.5 A resolution of a fragment of the integrase of Rous sarcoma virus (residues 49-286) containing both the conserved catalytic domain and a modulatory DNA-binding domain (C domain). The catalytic domains form a symmetric dimer, but the C domains associate asymmetrically with each other and together adopt a canted conformation relative to the catalytic domains. A binding path for the viral cDNA is evident spanning both domain surfaces, allowing modeling of the larger integration complexes that are known to be active in vivo. The modeling suggests that formation of an integrase tetramer (a dimer of dimers) is necessary and sufficient for joining both viral cDNA ends at neighboring sites in the target DNA. The observed asymmetric arrangement of C domains suggests that they could form a rotationally symmetric tetramer that may be important for bridging integrase complexes at each cDNA end.

Pregnancy produces significant alterations in the posture of the pregnant woman; however, gait changes that occur during pregnancy have not been adequately evaluated in the literature. This prospective investigation of the footprints of 25 pregnant women from early pregnancy to just prior to parturition revealed a significant increase in the base of gait during walking. This change in gait function may be a compensatory mechanism to improve locomotor stability, and may have important implications for foot function and development of lower-extremity pathology in pregnant women.

Cell membrane fusion by human (HIV) and simian (SIV) immunodeficiency viruses is mediated by the envelope glycoproteins gp120 and gp41. Although the precise mechanism of the fusion process is unknown, the ectodomain of gp41 is thought to undergo dramatic rearrangement from its prefusogenic state. To elucidate this process further, the crystal structure of the SIV gp41 ectodomain (residues 27-149) was determined at 1.47 A resolution and is reported herein. It is the most accurate and complete structure of a retroviral gp41 ectodomain determined to date. The rod-like trimeric structure of SIV gp41 comprises three parallel N-terminal alpha-helices assembled as a coiled coil in the center with three antiparallel C-terminal alpha-helices packed on the outside connected by highly flexible loops. Portions of the loops in all three monomers are crystallographically disordered and could not be accurately modeled. The core of the structure is similar (but not identical) to those of smaller HIV/SIV gp41 segments previously determined by X-ray crystallography with root mean square deviations in main chain atoms of less than 1.0 A. The crystal structure differs more substantially from the reported NMR solution structure of the identical SIV construct. The mechanisms of viral fusion and the inhibition by peptides are discussed in the context of the three-dimensional structure.

Recombinant Rous sarcoma virus integrase cloned from the Prague A (PrA) virus strain was expressed in Escherichia coli. Here we report the detailed purification procedure resulting in an apparently homogeneous integrase. Recombinant PrA integrase was compared at both the protein structural and the catalytic levels to avian myeloblastosis virus integrase purified from virions. Both proteins exist minimally in a dimeric state at low nanomolar concentrations as analyzed by glycerol gradient sedimentation and protein crosslinking studies. Likewise, both proteins have similar specific activities for full-site (concerted integration reaction) and half-site strand transfer activities using linear 480-bp retrovirus-like donor substrates that contain wild-type or mutant termini. They respond similarly to high NaCl concentrations ( approximately 350 mM) as well as aprotic solvents for efficient full-site strand transfer. The data suggest that recombinant integrase proteins with physical and catalytic properties similar to the virion counterpart can be purified using these techniques and will faithfully and efficiently promote the full-site integration reaction in vitro., (Copyright 1998 Academic Press.)

Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a secreted serpin identified in extracellular matrixes. We show that PEDF extractions from the interphotoreceptor matrix are more efficient with increasing NaCl concentrations, indicating that ionic interactions mediate its association with this polyanionic matrix. We have used affinity chromatography and ultrafiltration to probe for direct binding of PEDF to glycosaminoglycans/polyanions. Correctly folded PEDF bound to immobilized heparin, chondroitin sulfate-A, -B, -C, and dextran sulfate columns and eluted from each with an increase in NaCl concentration. However, in the presence of urea, the protein lost its affinity for heparin. Binding of PEDF to heparan sulfate proteoglycan in solution was in a concentration-dependent fashion (half-maximal specific binding EC50 = 40 micrograms/mL) and was sensitive to increasing NaCl concentrations. The glycosaminoglycan-binding region was analyzed using chemical modification and limited proteolysis. PEDF chemically modified on lysine residues by biotinylation lost its capacity for interacting with heparin, implicating the involvement of PEDF lysine residues in heparin binding. Cleavage of the serpin-exposed loop with chymotrypsin did not affect the heparin-binding property. A limited proteolysis product containing residues 21-approximately 260 bound to heparin with similar affinity as the intact PEDF. Homology modeling of PEDF based on the X-ray crystal structures of antithrombin III and ovalbumin shows a region at the center of beta-sheet A-strands 2 and 3- and helix F that has a basic electrostatic surface potential and is densely populated with lysines exposed to the surface (K134, K137, K189, K191, H212, and K214) that are available to interact with various glycosaminoglycans/polyanions. This region represents a novel site for glycosaminoglycan binding in a serpin, which in PEDF, is distinct and nonoverlapping from the PEDF neurotrophic active region.

The adsorption specificity of bacteriophage T4 is determined by genes 12 and 37, encoding the short tail-fibers (STF) and the distal part of the long tail-fibers (LTF), respectively. Both are trimeric proteins with rod domains made up of similar tandem quasi-repeats, approximately 40 amino acids long. Their assembly requires the viral chaperones gp57A and gp38. Here we report that fusing fragments of gp12 and gp37 to another trimeric T4 fibrous protein, fibritin, facilitates correct assembly, thereby by-passing the chaperone requirement. Fibritin is an alpha-helical coiled coil protein whose C-terminal part (fibritin E, comprising the last 120 residues) has recently been solved to atomic resolution. Gp12 fragments of 109 and 70 amino acids, corresponding to three and two quasi-repeats respectively, were fused to the C-terminus of fibritin E. A similar chimera was designed for the last 63 residues of gp37, which contain four copies of the pentapeptide Gly-X-His-X-His and assume a narrow rigid structure in the LTF distal tip. Expressed from plasmids, all three chimeras form soluble trimers that are resistant to dissociation by SDS and digestion by trypsin, indicative of correct folding and oligomerization.

The U3 and U5 termini of linear retrovirus DNA contain imperfect inverted repeats that are necessary for the concerted insertion of the termini into the host chromosome by viral integrase. Avian myeloblastosis virus integrase can efficiently insert the termini of retrovirus-like DNA donor substrates (480 base pairs) by a concerted mechanism (full-site reaction) into circular target DNA in vitro. The specific activities of virion-derived avian myeloblastosis virus integrase and bacterial recombinant Rous sarcoma virus (Prague A strain) integrase (approximately 50 nM or less) appear similar upon catalyzing the full-site reaction with 3'-OH recessed wild type or mutant donor substrates. We examined the role of the three nonsymmetrical nucleotides located at the 5th, 8th, and 12th positions in the U3 and U5 15-base pair inverted repeats for their ability to modify the full-site and simultaneously, the half-site strand transfer reactions. Our data suggest that the nucleotide at the 5th position appears to be responsible for the 3-5-fold preference for wild type U3 ends over wild type U5 ends by integrase for concerted integration. Additional mutations at the 5th or 6th position, or both, of U3 or U5 termini significantly increased (approximately 3 fold) the full-site reactions of mutant donors over wild type donors.

The env gene of SIV and HIV-1 encodes a single glycoprotein gp 160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N-terminal fusion peptide, of gp41 from HIV-1 (residues 27-154) and SIV (residues 27-149) have been expressed in Escherichia coli. These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein. Purified proteins have a trimeric subunit composition and high alpha-helical content, consistent with the predicted coil-coil structure. SIV gp41 containing a double cysteine mutation was crystallized. The crystals are suitable for X-ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry.

Two high resolution crystal structures of cytosolic aspartate aminotransferase from pig heart provide additional insights into the stereochemical mechanism for ligand-induced conformational changes in this enzyme. Structures of the homodimeric native structure and its complex with the substrate analog 2-methylaspartate have been refined, respectively, with 1.74-A x-ray diffraction data to an R value of 0.170, and with 1.6-A data to an R value of 0.173. In the presence of 2-methylaspartate, one of the subunits (subunit 1) shows a ligand-induced conformational change that involves a large movement of the small domain (residues 12-49 and 327-412) to produce a "closed" conformation. No such transition is observed in the other subunit (subunit 2), because crystal lattice contacts lock it in an "open" conformation like that adopted by subunit 1 in the absence of substrate. By comparing the open and closed forms of cAspAT, we propose a stereochemical mechanism for the open-to-closed transition that involves the electrostatic neutralization of two active site arginine residues by the negative charges of the incoming substrate, a large change in the backbone (phi,psi) conformational angles of two key glycine residues, and the entropy-driven burial of a stretch of hydrophobic residues on the N-terminal helix. The calculated free energy for the burial of this "hydrophobic plug" appears to be sufficient to serve as the driving force for domain closure.

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.

Bacteriophage T4 RNase H is a 5' to 3' exonuclease that removes RNA primers from the lagging strand of the DNA replication fork and is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. The crystal structure of the full-length native form of T4 RNase H has been solved at 2.06 angstroms resolution in the presence of Mg2+ but in the absence of nucleic acids. The most conserved residues are clustered together in a large cleft with two Mg2+ in the proposed active site. This structure suggests the way in which the widely separated conserved regions in the larger nucleotide excision repair proteins, such as human XPG, could assemble into a structure like that of the smaller replication nucleases.

The function of a flexible loop (loop 6) in the alpha-subunit from the tryptophan synthase alpha 2 beta 2 bienzyme complex has been investigated utilizing rapid-scanning (RSSF) and single-wavelength (SWSF) stopped-flow spectroscopies. Loop 6 is an extended sequence of residues which connects beta-strand 6 with alpha-helix 6 in the beta/alpha-barrel fold of the alpha-subunit. Substitution of Leu for Arg179 near the base of loop 6 does not significantly affect either the association of the alpha- and beta-subunits to form the bienzyme complex or the kinetics of the reaction of indole with L-serine (L-Ser) to form L-tryptophan (L-Trp), the process catalyzed by the wild-type beta-subunit [Kawasaki, H., Bauerle, R., Zon, G., Ahmed, S., & Miles, E. W. (1987) J. Biol. Chem. 262, 10678-10683]. However, the alpha-subunit-specific ligand glycerol phosphate (GP), which is an inhibitor of the wild-type beta-reaction, is a much less effective inhibitor of the alpha R179L-catalyzed beta-reaction. Equilibrium titration studies show that the affinity of GP for the alpha-site when either L-Ser or glycine is bound at the beta-site has been reduced by nearly 100- and 200-fold, respectively. SWSF analysis of the reaction of IGP and L-Ser to form L-Trp catalyzed by the bienzyme complex revealed a 15-fold reduction in the binding affinity of the alpha-site substrate 3-indole-D-glycerol 3'-phosphate (IGP) in the reaction catalyzed by the alpha R179L mutant as compared to the wild-type enzyme. These studies show that loop 6 is important both for ligand binding to the alpha-site and for the ligand-induced conformational transition of the alpha-subunit from an "open" to a "closed" structure. Modeling studies, based on extensive structural homology of the alpha-subunit with the glycolytic enzyme triosephosphate isomerase (TIM), predict that closure of loop 6 induced by ligand binding at the alpha-active site would effectively sequester the bound substrate from the solvent and trap indole, produced from the cleavage of IGP, within the confines of the bienzyme complex. This conformational transition would promote the diffusion of indole to the beta-active site via the interconnecting tunnel and would help ensure the close coordination of alpha- and beta-subunit catalytic activities.

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction. However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., Brzović, P., Drewe, W.F., Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an "open" to a "closed" structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open to the closed form.

Three successive steps in tryptophan biosynthesis are catalyzed by single-domain proteins, each folded as a parallel beta/alpha-barrel, as observed in the crystal structures of the bienzyme (phosphoribosyl)-anthranilate isomerase:indoleglycerolphosphate synthase from Escherichia coli [Priestle, J.P., Grütter, M. G., White, J. L., Vincent, M. G., Kania, M., Wilson, E., Jardetzky, T. S., Kirschner, K., & Jansonius, J. N. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5690-5694] and the alpha-subunit of the tetrameric bienzyme tryptophan synthase from Salmonella typhimurium [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Recent refinement of the crystal structures of these enzymes at atomic resolution revealed that they contain a common phosphate group binding site in the beta/alpha-barrel, created by residues of the loop between beta-strand 7 and alpha-helix 7 and the N-terminus of an additional helix 8'. The close similarities of their beta/alpha-barrel structures permitted the alignment of 50-75% of their respective amino acid sequences. Considerable sequence similarity was detected in the regions spanning the phosphate binding sites, whereas the percentage of identical residues was barely significant for the remaining parts of the enzymes. These observations suggest divergent evolution of these three beta/alpha-barrel enzymes involved in tryptophan biosynthesis. The same phosphate binding site was also observed in six other beta/alpha-barrel enzymes that are functionally unrelated to those involved in tryptophan biosynthesis: triosephosphate isomerase, ribulose-1,5-bisphosphate carboxylase/oxygenase, glycolate oxidase, flavocytochrome b2, trimethylamine dehydrogenase, and tentatively also fructosebisphosphate aldolase.(ABSTRACT TRUNCATED AT 250 WORDS)

The bifunctional tryptophan synthase alpha 2 beta 2 complex that catalyzes the final two reactions in tryptophan biosynthesis is a classic example of a multienzyme complex that "channels" a metabolic intermediate (indole) between two active sites. The three-dimensional structure of the alpha 2 beta 2 complex from Salmonella typhimurium reveals that the four polypeptide subunits are arranged in an extended alpha beta beta alpha order forming a complex 150 A long. The active sites of the neighboring alpha and beta subunits are separated by about 30 A and appear to be connected by a tunnel, which may facilitate the intramolecular transfer of indole. The active site of the alpha subunit, which is centrally located near one end of an eight-fold alpha/beta barrel structure, contains the sites of most of the missense mutations which were identified as key residues by Yanofsky and colleagues in early genetic studies. Site-directed mutagenesis is being used to replace residues found in the active sites of the alpha and beta subunits in order to probe the mechanism of catalysis. Recombinant DNA technology should also be useful in analyzing protein-protein interaction, protein folding and the channeling phenomenon.

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)

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.

Our studies, which are aimed at understanding the catalytic mechanism of the alpha subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to explore the functional roles of aspartic acid 60, tyrosine 175, and glycine 211. These residues are located close to the substrate binding site of the alpha subunit in the three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Our finding that replacement of aspartic acid 60 by asparagine, alanine, or tyrosine results in complete loss of activity in the reaction catalyzed by the alpha subunit supports a catalytic role for aspartic acid 60. Since the mutant form with glutamic acid at position 60 has partial activity, glutamic acid 60 may serve as an alternative catalytic base. The mutant form in which tyrosine 175 is replaced by phenylalanine has substantial activity; thus the phenolic hydroxyl of tyrosine 175 is not essential for catalysis or substrate binding. Yanofsky and colleagues have identified many missense mutant forms of the alpha subunit of tryptophan synthase from Escherichia coli. Two of these inactive mutant forms had either tyrosine 175 replaced by cysteine or glycine 211 replaced by glutamic acid. Surprisingly, a second-site revertant which contained both of these amino acid changes was partially active. These results indicated that the second mutation must compensate in some way for the first. We now extend the studies of the effects of specific amino acid replacements at positions 175 and 211 by two techniques: 1) characterization of several mutant forms of the alpha subunit from S. typhimurium prepared by site-directed mutagenesis and 2) computer graphics modeling of the substrate binding site of the alpha subunit using the x-ray coordinates of the wild type alpha 2 beta 2 complex from S. typhimurium. We conclude that the restoration of alpha subunit activity in the doubly altered second-site revertant results from restoration of the proper geometry of the substrate binding site.

Absorption spectra of large, well-formed crystals of cytosolic aspartate aminotransferase have been recorded using plane polarized light. Making use of measurements of crystal thickness we have calculated extinction coefficients with the electric vector of the light parallel to both the a and c axes of the crystals of the enzyme in space group P2(1)2(1)2(1). The spectra have been resolved into components with lognormal distribution curves and the resulting integrated intensities have been used to calculate the c/a polarization ratios for the absorption bands of the bound co-enzyme pyridoxal 5'-phosphate. From the polarization ratio and the co-ordinates of the co-enzyme ring atoms, provided by X-ray crystallography, we have assigned principal molecular directions of the transition dipole moment within the plane of the co-enzyme ring. Of two possible orientations, only one predicts the correct crystal extinction coefficients for the 436 nm band. In this orientation, when viewed from the B face of the ring (i.e. looking into the active site of the enzyme), the transition moment is related to the N-1-C-4 axis of the ring by counterclockwise rotation by 27 degrees. A tentative assignment of the principal molecular directions of the transition moment has also been made for the 368 nm band of the high pH form of the enzyme. In each case, the plane of the co-enzyme ring was located from the atomic co-ordinates of the ring atoms and of those atoms attached directly to the ring. The projection of the N-1 to C-4 axis on to this plane was used to evaluate the orientation of the transition moment, which was presumed to lie precisely within the plane of the ring. We have tilted this plane systematically to evaluate the error in transition moment direction resulting from uncertainties in the atomic co-ordinates. When 2-methylaspartate is diffused into the crystals if forms a Schiff base with the co-enzyme in which the ring has tilted about 32 degrees from its original position and the polarization ratio of the 436 nm band drops from 1.6 in the free enzyme to about 0.38. On the assumption that the orientation of the transition moment within the co-enzyme does not change during this rotation, this value of the polarization ratio is within experimental error of that predicted from X-ray structures on the two forms. The 2-methylaspartate binds only to subunit 1.(ABSTRACT TRUNCATED AT 250 WORDS)