Nine hydrophobic side chains are key determinants of the thermodynamic stability and oligomerization status of tumour suppressor p53 tetramerization domain.

The contribution of almost each amino acid side chain to the thermodynamic stability of the tetramerization domain (residues 326-353) of human p53 has been quantitated using 25 mutants with single-residue truncations to alanine (or glycine). Truncation of either Leu344 or Leu348 buried at the tetramer interface, but not of any other residue, led to the formation of dimers of moderate stability (8-9 kcal/mol of dimer) instead of tetramers. One-third of the substitutions were moderately destabilizing (<3.9 kcal/mol of tetramer). Truncations of Arg333, Asn345 or Glu349 involved in intermonomer hydrogen bonds, Ala347 at the tetramer interface or Thr329 were more destabilizing (4.1-5.7 kcal/mol). Strongly destabilizing (8.8- 11.7 kcal/mol) substitutions included those of Met340 at the tetramer interface and Phe328, Arg337 and Phe338 involved peripherally in the hydrophobic core. Truncation of any of the three residues involved centrally in the hydrophobic core of each primary dimer either prevented folding (Ile332) or allowed folding only at high protein concentration or low temperature (Leu330 and Phe341). Nine hydrophobic residues per monomer constitute critical determinants for the stability and oligomerization status of this p53 domain.     

Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F₁ with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F₁ sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β_DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β_E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.     

Hydrophobic interactions in the hinge domain of DNA polymerase beta are important but not sufficient for maintaining fidelity of DNA synthesis

We previously described a general mutator form of mammalian DNA polymerase beta containing a cysteine substitution for tyrosine 265. Residue 265 localizes to a hydrophobic hinge region predicted to mediate a polymerase conformational change that may aid in nucleotide selectivity. In this study we tested the hypothesis that van der Waals and hydrophobic contacts between Y265 and neighboring residues are important for DNA synthesis fidelity and catalysis, by altering interactions in the hinge domain via substitution at position 265. Consistent with the importance of hydrophobic interactions, we found that phenylalanine, leucine, and tryptophan substitutions did not alter significantly the steady-state catalytic efficiency of DNA synthesis, relative to wild type, while the polar serine substitution decreased catalytic efficiency 6-fold. However, we found that all substitutions other than phenylalanine increased the error frequency, relative to wild type, in the order serine > tryptophan = leucine. Therefore, maintenance of the hydrophobicity of residue 265 was not sufficient for maintaining fidelity of DNA synthesis. We conclude that while hydrophobic interactions in the hinge domain are important for fidelity, additional factors such as electrostatic and van der Waals interactions contributed by the tyrosine 265 aromatic ring are required to retain wild-type fidelity.     

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase has been determined at 2.7 A resolution. Its first 120 amino acids form a central five-stranded, beta-pleated sheet surrounded by five alpha helices. In one of the four dyad-related dimers, the two active site Ser-10 residues are 19 A apart, perhaps close enough to contact and become covalently linked to the DNA at the recombination site. This dimer also forms the only closely packed tetramer found in the crystal. The subunit interface at a second dyad-related dimer is more extensive and more highly conserved among the homologous recombinases; however, its active site Ser-10 residues are more than 30 A apart. Side chains, identified by mutations that eliminate catalysis but not DNA binding, are located on the subunit surface near the active site serine and at the interface between a third dyad-related pair of subunits of the tetramer.     

The dihedral symmetry of the p53 tetramerization domain mandates a conformational switch upon DNA binding.

The p53 tumor suppressor forms stable tetramers, whose DNA binding activity is allosterically regulated. The tetramerization domain is contained within the C-terminus (residues 323-355) and its three-dimensional structure exhibits dihedral symmetry, such that a p53 tetramer can be considered a dimer of dimers. Under conditions where monomeric p53 fails to bind DNA, we studied the effects of p53 C-terminal mutations on DNA binding. Residues 322-355 were sufficient to drive DNA binding of p53 as a tetramer. Within this region residues predicted by the three-dimensional structure to stabilize tetramerization, such as Arg337 and Phe341, were critical for DNA binding. Furthermore, substitution of Leu344 caused p53 to dissociate into DNA binding-competent dimers, consistent with the location of this residue at the dimer-dimer interface. The p53 DNA site contains two inverted repeats juxtaposed to a second pair of inverted repeats. Thus, the four repeats exhibit cyclic-translation symmetry and cannot be recognized simultaneously by four dihedrally symmetric p53 DNA binding domains. The discrepancy may be resolved by flexible linkers between the p53 DNA binding and tetramerization domains. When these linkers were deleted p53 exhibited novel DNA binding properties consistent with an inability to recognize four contiguous DNA repeats. Allosteric regulation of p53 DNA binding may involve repositioning the DNA binding domains from a dihedrally symmetric state to a DNA-bound asymmetric state.     

Hydrophobic side-chain size is a determinant of the three-dimensional structure of the p53 oligomerization domain.

The p53 tumor suppressor oligomerization domain, a dimer of two primary dimers, is an independently folding domain whose subunits consist of a beta-strand, a tight turn and an alpha-helix. To evaluate the effect of hydrophobic side-chains on three-dimensional structure, we substituted residues Phe341 and Leu344 in the alpha-helix with other hydrophobic amino acids. Substitutions that resulted in residue 341 having a smaller side-chain than residue 344 switched the stoichiometry of the domain from tetrameric to dimeric. The three-dimensional structure of one such dimer was determined by multidimensional NMR spectroscopy. When compared with the primary dimer of the wild-type p53 oligomerization domain, the mutant dimer showed a switch in alpha-helical packing from anti-parallel to parallel and rotation of the alpha-helices relative to the beta-strands. Hydrophobic side-chain size is therefore an important determinant of a protein fold.     

The Hin recombinase assembles a tetrameric protein swivel that exchanges DNA strands

Most site-specific recombinases can be grouped into two structurally and mechanistically different classes. Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements. Here we use site-directed crosslinking to investigate the conformational changes that accompany the formation of the synaptic complex and the exchange of DNA strands by the Hin serine recombinase. Efficient crosslinking between residues corresponding to the ‘D-helix’ region provides the first experimental evidence for interactions between synapsed subunits within this region and distinguishes between different tetrameric conformers that have been observed in crystal structures of related serine recombinases. Crosslinking profiles between cysteines introduced over the 35 residue E-helix region that constitutes most of the proposed rotating interface both support the long helical structure of the region and provide strong experimental support for a subunit rotation mechanism that mediates DNA exchange.     

Regulatory mutations in Sin recombinase support a structure-based model of the synaptosome

The resolvase Sin regulates DNA strand exchange by assembling an elaborate interwound synaptosome containing catalytic and regulatory Sin tetramers, and an architectural DNA-bending protein. The crystal structure of the regulatory tetramer was recently solved, providing new insights into the structural basis for regulation. Here we describe the selection and characterization of two classes of Sin mutations that, respectively, bypass or disrupt the functions of the regulatory tetramer. Activating mutations, which allow the catalytic tetramer to assemble and function independently at site I (the crossover site), were found at ∼20% of residues in the N-terminal domain. The most strongly activating mutation (Q115R) stabilized a catalytically active synaptic tetramer in vitro . The positions of these mutations suggest that they act by destabilizing the conformation of the ground-state site I-bound dimers, or by stabilizing the altered conformation of the active catalytic tetramer. Mutations that block activation by the regulatory tetramer mapped to just two residues, F52 and R54, supporting a functional role for a previously reported crystallographic dimer–dimer interface. We suggest how F52/R54 contacts between regulatory and catalytic subunits might promote assembly of the active catalytic tetramer within the synaptosome.     

DNA sequence analysis of the oli1 gene reveals amino acid changes in mitochondrial ATPase subunit 9 from oligomycin-resistant mutants of Saccharomyces cerevisiae

The nucleotide sequence of the oli1 gene encoding mitochondrial ATPase subunit 9 (76 amino acids) has been determined for five oligomycin-resistant mutants of Saccharomyces cerevisiae. Three of the mutations affect amino acids in the vicinity of the glutamic acid residue 59 at which dicylohexyl carbodiimide binds. Two other mutations lead to substitution of amino acid 23, which would lie very close to residue 59 in the folded hairpin conformation that this protein is thought to adopt in the inner mitochondrial membrane. The apposition of residues 23 and those adjacent to residue 59, lying respectively in the two hydrophobic membrane-spanning arms of subunit 9, is considered to constitute an oligomycin-binding domain. By consideration of the amino acid substitutions in those mutants cross-resistant to venturicidin, a domain of resistance for venturicidin is defined to lie within the oligomycin-binding domain, also centered on residues 23 and 59. These data also clarify the genetic recombination behaviour of alleles previously defined to form part of the oli3 locus (mutants characterized by resistance to both oligomycin and venturicidin) together with alleles defined to form part of the oli1 locus (mutants not cross-resistant to venturicidin). The oli1 and oli3 loci can now be seen to form two overlapping extended groups within the oli1 gene, with sequenced oli3 mutations being as far apart as 125 nucleotides within the subunit 9 coding region of 231 nucleotides.     

Roles of the P1, P2, and P3 residues in determining inhibitory specificity of kallistatin toward human tissue kallikrein

Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386-388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys >/= P1 Tyr > P1 Leu >/= P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr(99) and Trp(219) of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.     

Intrasubunit and intersubunit interactions controlling assembly of active synaptic complexes during Hin-catalyzed DNA recombination

Serine recombinases, which generate double-strand breaks in DNA, must be carefully regulated to ensure that chemically active DNA complexes are assembled correctly. In the Hin-catalyzed site-specific DNA inversion reaction, two inversely oriented recombination sites on the same DNA molecule assemble into a synaptic complex that uniquely generates inversion products. The Fis-bound recombinational enhancer, together with topological constraints directed by DNA supercoiling, functions to regulate Hin synaptic complex formation and activity. We have isolated a collection of gain-of-function mutants in 22 positions within the catalytic and oligomerization domains of Hin using two genetic screens and by site-directed mutagenesis. One genetic screen measured recombination in the absence of Fis and the other assessed SOS induction as a readout of increased DNA cleavage. These mutations, together with molecular modeling, identify important sites of dynamic intrasubunit and intersubunit interactions that regulate assembly of the active tetrameric recombination complex. Of particular interest are interactions between the oligomerization helix (helix E) and the catalytic domain of the same subunit that function to hold the dimer in an inactive state in the absence of the Fis/enhancer system. Among these is a relay involving a triad of phenylalanines that are proposed to switch positions during the transition from dimers to the catalytically active tetramer. Novel Hin mutants that generate synaptic complexes that are blocked at steps prior to DNA cleavage are also described.     

The structural basis for enhanced stability and reduced DNA binding seen in engineered second-generation Cro monomers and dimers

It was previously shown that the Cro repressor from phage lambda, which is a dimer, can be converted into a stable monomer by a five-amino acid insertion. Phe58 is the key residue involved in this transition, switching from interactions which stabilize the dimer to those which stabilize the monomer. Structural studies, however, suggested that Phe58 did not penetrate into the core of the monomer as well as it did into the native dimer. This was strongly supported by the finding that certain core-repacking mutations, including in particular, Phe58-->Trp, increased the stability of the monomer. Unexpectedly, the same substitution also increased the stability of the native dimer. At the same time it decreased the affinity of the dimer for operator DNA. Here we describe the crystal structures of the Cro F58W mutant, both as the monomer and as the dimer. The F58W monomer crystallized in a form different from that of the original monomer. In contrast to that structure, which resembled the DNA-bound form of Cro, the F58W monomer is closer in structure to wild-type (i.e. non-bound) Cro. The F58W dimer also crystallizes in a form different from the native dimer but has a remarkably similar overall structure which tends to confirm the large changes in conformation of Cro on binding DNA. Introduction of Trp58 perturbs the position occupied by the side-chain of Arg38, a DNA-contact residue, providing a structural explanation for the reduction in DNA-binding affinity.The improved thermal stability is seen to be due to the enhanced solvent transfer free energy of Trp58 relative to Phe58, supplemented in the dimer structure, although not the monomer, by a reduction in volume of internal cavities.     

Conformational shifts propagate from the oligomerization domain of p53 to its tetrameric DNA binding domain and restore DNA binding to select p53 mutants.

p53 is a conformationally flexible sequence-specific DNA binding protein mutated in many human tumors. To understand why the mutant p53 proteins associated with human tumors fail to bind DNA, we mapped the DNA binding domain of wild-type p53 and examined its regulation by changes in the protein conformation. Using site-directed mutagenesis, residues 90-286 of mouse p53 were shown to form the sequence-specific DNA binding domain. Two highly conserved regions within this domain, regions IV and V, were implicated in contacting DNA. Wild-type p53 bound DNA as a tetramer, each subunit recognizing five nucleotides of the 20 nucleotide-long DNA site. Conformational shifts of the oligomerization domain propagated to the tetrameric DNA binding domain, regulating DNA binding activity, but did not affect the subunit stoichiometry of wild-type p53 oligomers. Interestingly, conformational shifts could also be propagated within certain p53 mutants, rescuing DNA binding. One of these mutants was the mouse equivalent of human histidine 273, which is frequently associated with human tumors.     

Chemical shift mapping of gammadelta resolvase dimer and activated tetramer: mechanistic implications for DNA strand exchange

Several static structural models exist for gammadelta resolvase, a self-coded DNA recombinase of the gammadelta transposon. While these reports are invaluable to formulation of a mechanistic hypothesis for DNA strand exchange, several questions remain. Foremost among them concerns the protomer structural dynamics within the protein/DNA synaptosome. Solution NMR chemical shift assignments have been made for truncated variants of the natural wild-type dimer, which is inactive without the full synaptosome structure, and a mutationally activated tetramer. Of the 134 residues, backbone (1)H, (15)N, and (13)Calpha assignments are made for 121-124 residues in the dimer, but only 76-80 residues of the tetramer. These assignment differences are interpreted by comparison to X-ray diffraction models of the recombinase dimer and tetramer. Inspection of intramolecular and intermolecular structural variation between these models suggests a correspondence between sequence regions at subunit interfaces unique to tetramer, and the regions that can be sequentially assigned in the dimer but not the tetramer. The loss of sequential context for assignment is suggestive of stochastic fluctuation between structural states involving protomer-protomer interactions exclusive to the activated tetrameric state, and may be indicative of dynamics which pertain to the recombinase mechanism.     

Interdomain side-chain interactions in human gammaD crystallin influencing folding and stability

Human gammaD crystallin (HgammaD-Crys) is a two domain, beta-sheet eye lens protein that must remain soluble throughout life for lens transparency. Single amino acid substitutions of HgammaD-Crys are associated with juvenile-onset cataracts. Features of the interface between the two domains conserved among gamma-crystallins are a central six-residue hydrophobic cluster, and two pairs of interacting residues flanking the cluster. In HgammaD-Crys these pairs are Gln54/Gln143 and Arg79/Met147. We previously reported contributions of the hydrophobic cluster residues to protein stability. In this study alanine substitutions of the flanking residue pairs were constructed and analyzed. Equilibrium unfolding/refolding experiments at 37 degrees C revealed a plateau in the unfolding/refolding transitions, suggesting population of a partially folded intermediate with a folded C-terminal domain (C-td) and unfolded N-terminal domain (N-td). The N-td was destabilized by substituting residues from both domains. In contrast, the C-td was not significantly affected by substitutions of either domain. Refolding rates of the N-td were significantly decreased for mutants of either domain. In contrast, refolding rates of the C-td were similar to wild type for mutants of either domain. Therefore, domain interface residues of the folded C-td probably nucleate refolding of the N-td. We suggest that these residues stabilize the native state by shielding the central hydrophobic cluster from solvent. Glutamine and methionine side chains are among the residues covalently damaged in aged and cataractous lenses. Such damage may generate partially unfolded, aggregation- prone conformations of HgammaD-Crys that could be significant in cataract.     

How p53 binds DNA as a tetramer.

The p53 tumor suppressor protein is a tetramer that binds sequence-specifically to a DNA consensus sequence consisting of two consecutive half-sites, with each half-site being formed by two head-to-head quarter-sites (--><-- --><--). Each p53 subunit binds to one quarter-site, resulting in all four DNA quarter-sites being occupied by one p53 tetramer. The tetramerization domain forms a symmetric dimer of dimers, and two contrasting models have the two DNA-binding domains of each dimer bound to either consecutive or alternating quarter-sites. We show here that the two monomers within a dimer bind to a half-site (two consecutive quarter-sites), but not to separated (alternating) quarter-sites. Tetramers bind similarly, with the two dimers within each tetramer binding to pairs of half-sites. Although one dimer within the tetramer is sufficient for binding to one half-site in DNA, concurrent interaction of the second dimer with a second half-site in DNA drastically enhances binding affinity (at least 50-fold). This cooperative dimer-dimer interaction occurs independently of tetramerization and is a primary mechanism responsible for the stabilization of p53 DNA binding. Based on these findings, we present a model of p53 binding to the consensus sequence, with the tetramer binding DNA as a pair of clamps.     

Role of residue Phe225 in the cofactor-mediated, allosteric regulation of the serine protease coagulation factor VIIa

Functional regulation by cofactors is fundamentally important for the highly ordered, consecutive activation of the coagulation cascade. The initiating protease of the coagulation system, factor VIIa (VIIa), retains zymogen-like features after proteolytic cleavage of the activating Arg(15)-Ile(16) peptide bond and requires the binding of the cofactor tissue factor (TF) to stabilize the protease domain in an active enzyme conformation. Structural comparison of TF-bound and free VIIa failed to provide a conclusive mechanism for this catalytic activation. This study provides novel insight into the cofactor-dependent regulation of VIIa by demonstrating that the side chain of Phe(225), an aromatic residue that is common to allosterically regulated serine proteases, is necessary for optimal TF-mediated activation of VIIa's catalytic function. However, mutation of Phe(225) did not abolish the cofactor-induced stabilization of the Ile(16)-Asp(194) salt bridge, previously considered the primary switch mechanism for activating VIIa. Moreover, mutation of other residue side chains in the VIIa protease domain resulted in a reduced level of or no stabilization of the amino-terminal insertion site upon TF binding, with little or no effect on the TF-mediated enhancement of catalysis. This study thus establishes a crucial role for the aromatic Phe(225) residue position in the allosteric network that transmits the activating switch from the cofactor interface to the catalytic cleft, providing insight into the highly specific conformational linkages that regulate serine protease function.     

Chemical shift mapping of γδ resolvase dimer and activated tetramer: Mechanistic implications for DNA strand exchange

Several static structural models exist for γδ resolvase, a self-coded DNA recombinase of the γδ transposon. While these reports are invaluable to formulation of a mechanistic hypothesis for DNA strand exchange, several questions remain. Foremost among them concerns the protomer structural dynamics within the protein/DNA synaptosome. Solution NMR chemical shift assignments have been made for truncated variants of the natural wild-type dimer, which is inactive without the full synaptosome structure, and a mutationally activated tetramer. Of the 134 residues, backbone ¹H, ¹⁵N, and ¹³Cα assignments are made for 121–124 residues in the dimer, but only 76–80 residues of the tetramer. These assignment differences are interpreted by comparison to X-ray diffraction models of the recombinase dimer and tetramer. Inspection of intramolecular and intermolecular structural variation between these models suggests a correspondence between sequence regions at subunit interfaces unique to tetramer, and the regions that can be sequentially assigned in the dimer but not the tetramer. The loss of sequential context for assignment is suggestive of stochastic fluctuation between structural states involving protomer–protomer interactions exclusive to the activated tetrameric state, and may be indicative of dynamics which pertain to the recombinase mechanism.     

Ydj1p二聚体中β14~β15与domain-III分离的拉伸分子动力学模拟研究

分子伴侣Hsp40是一种以二聚体的形式调控非天然多肽折叠的热激蛋白。本文通过拉伸分子动力学研究了酵母Hsp40家族成员Ydj1p二聚体中β14-β15与domain-Ⅲ的分离过程,深入探讨了影响Ydj1p二聚体稳定性的重要残基和相互作用力。研究表明,残基Thr366、Asp368、Cys370、Leu372和Phe375在Ydj1P二聚体的形成过程中发挥着重要的作用。其中,β14-β15中的残基Thr366和Asp368分别通过与domain-Ⅲ内的残基Asp291、Trp292和Trp292、Lys294之间形成的氢键,Asp368通过与domain-Ⅲ内的残基Lys314形成盐桥,Cys370、Leu372和Phe375则是通过与domain-Ⅲ形成疏水作用力来稳定Ydj1p二聚体结构。     

Enzymatic properties of mutant Escherichia coli tryptophan synthase alpha-subunits

Thirty-nine mutant tryptophan synthase alpha subunits have been purified and analyzed (in the presence of the beta 2-subunit) for their enzymatic (kcat, Km) behavior in the reactions catalyzed by the alpha 2.beta 2 complex, the fully constituted form of this enzyme. The mutant alpha subunits, obtained by in vitro random, saturation mutagenesis of the encoding trpA gene, contain single amino acid substitutions at sites within the first 121 residues of the alpha polypeptide. Four categories of altered residues have been tentatively assigned roles in the catalytic functions of this enzyme: 1) catalytic residues (Glu49 and Asp60); 2) residues involved in substrate binding or orientation (Phe22, Thr63, Gln65, Tyr102, and Leu105); 3) residues involved in alpha.beta subunit interactions (Gly51, Pro53, Asp56, Asp60, Pro62, Ala67, Phe72, Thr77, Pro78, Tyr102, Asn104, Leu105, and Asn108); and 4) residues with no apparent catalytic roles. Catalytic residue alterations result in no detectable activity in the alpha-subunit specific reactions. Substrate binding/orientation roles are detected enzymatically primarily as rate defects; alterations only at Tyr102 result in apparent Km effects. alpha.beta interaction roles are detected as rate defects in all tryptophan synthase reactions plus Km increases for the alpha-subunit substrate, indole-3-glycerol phosphate, only when L-serine is present at the beta 2-subunit active site. A substitution at only one site, Asn104, appears to be unique in its potential effect on intersubunit channeling of indole, the product of the alpha-subunit specific reaction, to the beta 2-subunit active site.