Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10(-15) M.

We have designed a heterodimerizing leucine zipper system to target a radionuclide to prelocalized noninternalizing tumor-specific antibodies. The modular nature of the leucine zipper allows us to iteratively use design rules to achieve specific homodimer and heterodimer affinities. We present circular-dichroism thermal denaturation measurements on four pairs of heterodimerizing leucine zippers. These peptides are 47 amino acids long and contain four or five pairs of electrostatically attractive g <--> e' (i, i' +5) interhelical heterodimeric interactions. The most stable heterodimer consists of an acidic leucine zipper and a basic leucine zipper that melt as homodimers in the micro (T(m) = 28 degrees C) or nanomolar (T(m) = 40 degrees C) range, respectively, but heterodimerize with a T(m) >90 degrees C, calculated to represent femtamolar affinities. Modifications to this pair of acidic and basic zippers, designed to destabilize homodimerization, resulted in peptides that are unstructured monomers at 4 microM and 6 degrees C but that heterodimerize with a T(m) = 74 degrees C or K(d(37)) = 1.1 x 10(-11) M. A third heterodimerizing pair was designed to have a more neutral isoelectric focusing point (pI) and formed a heterodimer with T(m) = 73 degrees C. We can tailor this heterodimerizing system to achieve pharmacokinetics aimed at optimizing targeted killing of cancer cells.

Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system produced by substituting alanine or glutamate for the active-site histidine 189: implications for phosphorylation effects.

The amino terminal domain of enzyme I (residues 1-258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active-site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature-induced changes in ellipticity at 222 nm for monomeric wild-type and mutant EIN proteins indicate two-state unfolding. For EIN proteins in 10 mM K-phosphate (and 100 mM KCl) at pH 7.5, deltaH approximately 140 +/- 10 (160) kcal mol(-1) and deltaCp approximately 2.7 (3.3) kcal K(-1) mol(-1). Transition temperatures (Tm) are 57 (59), 55 (58), and 53 (56) degrees C for wild-type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho-His189, phospho-His189, and H189 substitutions of EIN at pH 7.5 is: His > Ala > Glu > His-PO3(2-) due to differences in conformational entropy. Although H189E mutants have decreased Tm values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (Tm approximately 66-68 degrees C). This possibly arises from an ion pair interaction between the gamma-carboxyl of Glu189 and the epsilon-amino group of Lys69 in the docking region for the histidine-containing phosphocarrier protein HPr. However, the binding of HPr to wild-type and active-site mutants of EIN and EI is temperature-independent (entropically controlled) with about the same affinity constant at pH 7.5: K(A)' = 3 +/- 1 x 10(5) M(-1) for EIN and approximately 1.2 x 10(5) M(-1) for EI.

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3. The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.

Cellobiose-6-phosphate hydrolase (CelF) of Escherichia coli: characterization and assignment to the unusual family 4 of glycosylhydrolases.

The gene celF of the cryptic cel operon of Escherichia coli has been cloned, and the encoded 6-phospho-beta-glucosidase (cellobiose-6-phosphate [6P] hydrolase; CelF [EC 3.2.1.86]) has been expressed and purified in a catalytically active state. Among phospho-beta-glycosidases, CelF exhibits unique requirements for a divalent metal ion and NAD(+) for activity and, by sequence alignment, is assigned to family 4 of the glycosylhydrolase superfamily. CelF hydrolyzed a variety of P-beta-glucosides, including cellobiose-6P, salicin-6P, arbutin-6P, gentiobiose-6P, methyl-beta-glucoside-6P, and the chromogenic analog, p-nitrophenyl-beta-D-glucopyranoside-6P. In the absence of a metal ion and NAD(+), purified CelF was rapidly and irreversibly inactivated. The functional roles of the cofactors have not been established, but NAD(+) appears not to be a reactant and there is no evidence for reduction of the nucleotide during substrate cleavage. In solution, native CelF exists as a homotetramer (M(w), approximately 200,000) composed of noncovalently linked subunits, and this oligomeric structure is maintained independently of the presence or absence of a metal ion. The molecular weight of the CelF monomer (M(r), approximately 50,000), estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is in agreement with that calculated from the amino acid sequence of the polypeptide (450 residues; M(r) = 50,512). Comparative sequence alignments provide tentative identification of the NAD(+)-binding domain (residues 7 to 40) and catalytically important glutamyl residues (Glu(112) and Glu(356)) of CelF.

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at

The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD(H)-dependent 6-phospho-alpha-glucosidase. Assignment to family 4 of the glycosylhydrolase superfamily.

The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, Mr = 50,513) is a unique 6-phosphoryl-O-alpha-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-alpha-glucosidase) that requires both NAD(H) and divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) for activity. 6-Phospho-alpha-glucosidase (EC 3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the Mr of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site-directed mutagenesis shows that three acidic residues, Asp41, Glu111, and Glu359, are required for GlvA activity. Asp41 is located at the C terminus of a betaalphabeta fold that may constitute the dinucleotide binding domain of the protein. Glu111 and Glu359 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-alpha-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate. In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn2+ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer. By comparative sequence alignment with its homologs, the novel 6-phospho-alpha-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.

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

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

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

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

Thermal inactivation of tryptophan synthase. Stabilization by protein-protein interaction and protein-ligand interaction.

This study investigates effects of ligands on thermal inactivation of the tryptophan synthase alpha and beta 2 subunits alone and in the alpha 2 beta 2 complex. Addition of pyridoxal phosphate to the apo-beta 2 subunit increases the temperature of one-half inactivation (Ti) from 52 to 77 degrees C. Ligands that promote association of the alpha and holo-beta 2 subunits markedly stabilize the more temperature-labile alpha subunit in the alpha 2 beta 2 complex from irreversible thermal denaturation. The combination of a beta 2 subunit ligand (L-serine) with an alpha subunit ligand (alpha-glycerol 3-phosphate) raises the inactivation temperature (Ti) of the alpha subunit in the holo-alpha 2 beta 2 complex from 54 to 66 degrees C. In contrast, values of Ti for inactivation of the alpha and beta subunits in the holo-alpha 2 beta 2 complex are more similar to respective values for the isolated alpha subunit (50 degrees C) and holo-beta 2 subunit (77 degrees C). Surprisingly, the addition of L-serine results in a larger decrease in the Ti of the beta 2 subunit in the holo-alpha 2 beta 2 complex (78 degrees C-->64 degrees C) than in Ti of the holo-beta 2 subunit alone (77 degrees C-->71 degrees C). The observation that ligands have different effects on the isolated and associated subunits provides evidence that the alpha and beta 2 subunits do not fully dissociate during thermal inactivation of the alpha 2 beta 2 complex at pH 7.8 and at approximately 0.1 ionic strength. Our results demonstrate that linkage between protein-ligand interactions and protein-protein interactions affects the conformational stability of the tryptophan synthase alpha 2 beta 2 complex.

Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium.

To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual alpha and beta subunits. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.

Subunit communication in the tryptophan synthase alpha 2 beta 2 complex. Effects of beta subunit ligands on proteolytic cleavage of a flexible loop in the alpha subunit.

To probe the structural basis for ligand-mediated communication between the alpha and beta subunits in the tryptophan synthase alpha 2 beta 2 complex, we have determined the effects of ligands of the alpha and beta subunits on proteolysis of a flexible loop in the alpha subunit. We find that addition of a ligand of the beta subunit (L-serine, D-tryptophan, or L-tryptophan) in combination with a ligand of the alpha subunit (alpha-glycerol 3-phosphate) almost completely prevents the tryptic cleavage of the alpha subunit loop. Thus, the binding of a ligand to the beta-site affects the conformation of the alpha subunit 25-30 A distant.

Mechanism of mutual activation of the tryptophan synthase alpha and beta subunits. Analysis of the reaction specificity and substrate-induced inactivation of active site and tunnel mutants of the beta subunit.

The origin of reaction and substrate specificity and the control of activity by protein-protein interaction are investigated using the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. We have compared some spectroscopic and kinetic properties of the wild type beta subunit and five mutant forms of the beta subunit that have altered catalytic properties. These mutant enzymes, which were engineered by site-directed mutagenesis, have single amino acid replacements in either the active site or in the wall of a tunnel that extends from the active site of the alpha subunit to the active site of the beta subunit in the alpha 2 beta 2 complex. We find that the mutant alpha 2 beta 2 complexes have altered reaction and substrate specificity in beta-elimination and beta-replacement reactions with L-serine and with beta-chloro-L-alanine. Moreover, the mutant enzymes, unlike the wild type alpha 2 beta 2 complex, undergo irreversible substrate-induced inactivation. The mechanism of inactivation appears to be analogous to that first demonstrated by Metzler's group for inhibition of two other pyridoxal phosphate enzymes. Alkaline treatment of the inactivated enzyme yields apoenzyme and a previously described pyridoxal phosphate derivative. We demonstrate for the first time that enzymatic activity can be recovered by addition of pyridoxal phosphate following alkaline treatment. We conclude that the wild type and mutant alpha 2 beta 2 complexes differ in the way they process the amino acrylate intermediate. We suggest that the wild type beta subunit undergoes a conformational change upon association with the alpha subunit that alters the reaction specificity and that the mutant beta subunits do not undergo the same conformational change upon subunit association.

[Factors, determining the effectiveness of tryptophanase interaction with amino acids]. / Faktory, opredeliaiushchie éffektivnost' vzaimodeistviia triptofanazys aminokislotami.

Inhibition of tryptophanase-catalyzed decomposition of S-(o-nitrophenyl)-L-cysteine by a variety of amino acids has been investigated. For amino acids similar to the natural substrate and for those having minimal steric requirements for the side chain, the linear correlation exists between-RTlnKi and side chain hydrophobicity. L-ornithine and L-arginine are anomalously potent inhibitors taking into account low hydrophobicity of their side chains. This can be explained by an interaction between a positively charged group of the side chain of L-arginine or L-ornithine and a nucleophilic group of the active site. The comparison of affinity of tryptophanase for L-phenylalanine and L-homophenylalanine indicates that there is a special locus in the active site where aromatic groups are bound and oriented approximately parallel to the cofactor plane experiencing no steric hindrance. For a large number of amino acids the rates of the enzymic alpha-proton exchange in 2H2O are comparable with the rate of the reaction with L-tryptophan. Very low rate of alpha-proton exchange observed with L-alanine is an exception.

Tyrosine phenol-lyase from Citrobacter intermedius. Factors controlling substrate specificity.

L-Amino acids are competitive inhibitors of tyrosine phenol-lyase from Citrobacter intermedius. For non-branched amino acids the correlation exists between -RTlnKi and side-chain hydrophobicity. Aspartic and glutamic acids are anomalously potent inhibitors taking into account low hydrophobicity of their side chains. This suggests the presence of an electrophilic group in the active site which interacts with the terminal carboxylic group of aspartic or glutamic acids. Tyramine, beta-phenylethylamine and tryptamine do not display detectable inhibition. The esters and amides of aromatic L-amino acids, D-phenylalanine and D-tryptophan are competitive inhibitors. The enzymatic isotope exchange of the alpha-proton in 2H2O was observed only in the case of L-amino acids. For L-phenylalanine and L-tryptophan it was shown to proceed with complete retention of configuration. The substrate specificity of tyrosine phenol-lyase is controlled during the stage of phenol elimination. The OH group in the para position of the ring is necessary for this stage to proceed. The same stage is also sensitive to the steric parameters of the substituent in the ring which ensures the second factor of control. When all the requirements of substrate specificity are fulfilled (L-tyrosine, 3-fluoro-L-tyrosine), the 'key' phenol-elimination step is not the rate-limiting one, the reaction velocity being determined by the preceding alpha-proton abstraction.

[Interaction of tyrosine-phenol-lyase from Citrobacter intermedius with amino acids and their derivatives: factors determining the effectiveness of binding]. / Vzaimodeistvie tirozin-fenol-liazy iz Citrobacter intermedius s aminokislotami i ikh proizvodnymi: faktory, opredeliaiushchie éffektivnost' sviazyvaniia.

The inhibition by L-amino acids and their derivatives of tyrosine phenol-lyase is investigated. Tyramine, alpha-phenylethylamine and tryptamine have no detectable inhibition effect and hence are weakly bonded by an active site. The aromatic amino acid amides are competitive inhibitors but do not manifest an enzymatic isotope exchange of alpha-proton in D2O. Free amino acids however are competitive inhibitors and in the majority of cases exchange alpha-proton. The presence of COOH-group is therefore an important feature which determines the binding efficiency and causes the "active" conformation of the amino acid-PLP complex labelising alpha-proton. In the absence of functional and bulky groups in the amino acid side chain the hydrophobicity is found to be the main factor determining the binding efficiency. For these amino acids a correlation exists between-RTlnKi and side chain hydrophobicity. The amino acids bearing the bulky groups, i. e. valine, leucine and isoleucine have reduced binding efficiency. Lysine and arginine bearing positively charged functional groups possess no inhibition effect. Aspartic and glutamic acids are anomalously strong inhibitors taking into consideration low hydrophobicity of their side chains. One can assume that the electrophilic group able to interact with the terminal COO- -group of aspartic and glutamic acids is located in the active site of tyrosine phenollyase.

[Substrate specificity of tyrosine-phenol-lyase. Electron and steric control at the stage of aromatic moiety elimination]. / Substratnaia spetsifichnost' tirozin-fenol-liazy. Elektronnyi i stericheskii kontrol' na stadii otshchepleniia aromaticheskogo fragmenta.

We have investigated the electronic and steric effects of substituents in the aromatic moiety of the substrate on the two principal stages of the reaction catalyzed by tyrosine-phenol-lyase. The substrate specificity of the enzyme is controlled during the stage of elimination of the aromatic ring. The process may be formally considered as an electrophilic substitution in the aromatic nucleus and includes tautomerization of the phenol group into cyclohexadienone and subsequent beta-elimination with regeneration of aromaticity in the leaving group. The OH-group in the rho-position of the ring is the first necessary condition for the stage to proceed. The same stage is also sensitive to the steric parameters of the substituent in the ring which ensures the second factor of control. When the requirements of substrate specificity are fulfilled (L-tyrosine, 3-F-L-tyrosine) the "key" stage of elimination of phenol moiety is not the rate-limiting one, the velocity of the reaction being determined by the preceding stage of alpha-proton abstraction.