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K Kirschner

Publications and source records attributed to K Kirschner.

At least 37 records · Page 2Linked to original sources
PubMed1989

Correct folding of circularly permuted variants of a beta alpha barrel enzyme in vivo.

An important question in protein folding is whether the natural amino and carboxyl termini and the given order of secondary structure segments are critical to the stability and to the folding pathway of proteins. Here it is shown that two circularly permuted versions of the gene of a single-domain beta alpha barrel enzyme can be expressed in Escherichia coli. The variants are enzymically active and are practically indistinguishable from the original enzyme by several structural and spectroscopic criteria, despite the creation of new termini and the cleavage of a surface loop. This novel genetic approach should be useful for protein folding studies both in vitro and in vivo.

Aldose-Ketose Isomerases↗
PubMed1988

The role of the TRP1 gene in yeast tryptophan biosynthesis.

Transcription of the gene for phosphoribosyl-anthranilate isomerase (TRP1) from the TRP1 promoter is initiated only approximately half as frequently as, for example, from the TRP3 promoter, but TRP1 mRNA is approximately twice as stable as TRP3 mRNA. Therefore, the steady state amount of TRP1 mRNA in yeast cells, grown without amino acid limitation, is similar to the steady-state amount of TRP3 mRNA. The protein concentration of both enzymes in yeast cells is about the same, but the basal specific enzyme activity in permeabilized cells of the TRP1 gene product N-(5'-phosphoribosyl-1)-anthranilate isomerase is about 2-3 times higher than that of any of the other TRP enzymes. According to the kinetic parameters of the purified isomerase protein, the enzyme is more active than, for example, the purified TRP3 enzyme indoleglycerol-phosphate synthase. It is suggested that the TRP1 gene of Saccharomyces cerevisiae might be the result of a rearrangement event, separating the N-(5'-phosphoribosyl-1)-anthranilate isomerase domain from the indoleglycerol-phosphate synthase domain and putting the catalytically more active isomerase domain behind a weak and nonregulated constitutive promoter.

Aldose-Ketose Isomerases↗
PubMed1987

Prediction of secondary structure by evolutionary comparison: application to the alpha subunit of tryptophan synthase.

The amino acid sequences of the a subunits of tryptophan synthase from ten different microorganisms were aligned by standard procedures. The alpha helices, beta strands and turns of each sequence were predicted separately by two standard prediction algorithms and averaged at homologous sequence positions. Additional evidence for conserved secondary structure was derived from profiles of average hydropathy and chain flexibility values, leading to a joint prediction. There is good agreement between (1) predicted beta strands, maximal hydropathy and minimal flexibility, and (2) predicted loops, great chain flexibility, and protein segments that accept insertions of various lengths in individual sequences. The a subunit is predicted to have eight repeated beta-loop-alpha-loop motifs with an extra N-terminal alpha helix and an intercalated segment of highly conserved residues. This pattern suggests that the territory structure of the a subunit is an eightfold alpha/beta barrel. The distribution of conserved amino acid residues and published data on limited proteolysis, chemical modification, and mutagenesis are consistent with the alpha/beta barrel structure. Both the active site of the a subunit and the combining site for the beta 2 subunit are at the end of the barrel formed by the carboxyl-termini of the beta strands.

Amino Acid Sequence↗
PubMed1987

Three-dimensional structure of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli.

N-(5'-Phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli is a monomeric bifunctional enzyme of Mr 49,500 that catalyzes two sequential reactions in the biosynthesis of tryptophan. The three-dimensional structure of the enzyme has been determined at 2.8-A resolution by x-ray crystallography. The two catalytic activities reside on distinct functional domains of similar folding, that of an eightfold parallel beta-barrel with alpha-helices on the outside connecting the beta-strands. Both active sites were located with an iodinated substrate analogue and found to be in depressions on the surface of the domains created by the outward-curving loops between the carboxyl termini of the beta-sheet strands and the subsequent alpha-helices. They do not face each other, making "channeling" of the substrate between active sites virtually impossible. Despite the structural similarity of the two domains, no significant sequence homology was found when topologically equivalent residues were compared.

Aldose-Ketose Isomerases↗
PubMed1986

Identification of three sites of proteolytic cleavage in the hinge region between the two domains of the beta 2 subunit of tryptophan synthase of Escherichia coli or Salmonella typhimurium.

The beta 2 subunit of tryptophan synthase is composed of two independently folding domains connected by a hinge segment of the polypeptide that is particularly susceptible to limited proteolysis by trypsin [Högberg-Raibaud, A., & Goldberg, M. (1977) Biochemistry 16, 4014-4019]. Since tryptic cleavage in the hinge region inactivates the beta 2 subunit, the spatial relationship between the two domains is important for enzyme activity. However, it was not previously known whether inactivation results from cleavage of the chain or from the loss of internal fragment(s) subsequent to cleavage at two or more sites. We now report comparative studies of limited proteolysis by three proteinases: trypsin and endoproteinases Lys-C and Arg-C. Our key finding that endoproteinase Arg-C inactivates the beta 2 subunit by cleavage at a single site (Arg-275) demonstrates the important role of the hinge peptide for enzymatic activity. We have also identified the sites of cleavage and the time course of proteolysis by trypsin at Arg-275, Lys-283, and Lys-272 and by endoproteinase Lys-C at Lys-283 and Lys-272. Sodium dodecyl sulfate gel electrophoresis, Edman degradation, and carboxypeptidases B and Y have been used to identify the several fragments and peptides produced. Our finding that the beta 2 subunit and F1 fragments have a heterogeneous amino terminus (Met-1 or Thr-2) indicates that the amino-terminal methionine is incompletely removed during posttranslational modification. Our results show that Edman degradation can be effectively used with a protein of known sequence to analyze proteolytic digests that have at least four different amino-terminal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1985

The domain structure of tryptophan synthase. A neutron scattering study.

Tryptophan synthase from Escherichia coli is a complex of two alpha subunits and two beta subunits. Small-angle neutron scattering involving deuterium-labelled isomers revealed the quaternary structure of the enzyme at the level of the beta 2 subunit and the two structural domains P1 and P2 which constitute the alpha subunits. Within the alpha 2 beta 2 complex, the two alpha subunits are completely separated. They are situated on opposite sides of the beta 2 subunit. The most probable distance between the two alpha protomers is 10.5 +/- 1 nm; the nearest distance is 5.8 +/- 0.5 nm, and the largest distance is 13.5 +/- 0.5 nm. The two domains of the same alpha subunit are intimately juxtaposed. The distances between two like or unlike domains belonging to opposite alpha subunits are roughly equal. All domains exhibit about equal distances to the beta 2 subunit which is situated in the centre of the complex. Thus the cleft between P1 and P2, which probably contains the active site of the alpha subunit, makes intimate contact with the beta 2 subunit. Neutron scattering allows us to determine the shape of the beta 2 subunit within the complex. Comparison with the free dimer suggests a conformational change, upon assembly, from an elongated into a more compact form.

Deuterium↗
PubMed1985

Purification and characterization of the indole-3-glycerolphosphate synthase/anthranilate synthase complex of Saccharomyces cerevisiae.

The indole-3-glycerolphosphate synthase/anthranilate synthase complex from Saccharomyces cerevisiae was purified to apparent homogeneity. The native complex with Mr approximately equal to 130 000 consists of two different subunits, the TRP2 gene product with Mr = 64 000 and the TRP3 gene product with Mr = 58 000. The larger polypeptide was identified as anthranilate synthase and is active in vitro with ammonia as cosubstrate without need of complex formation. The smaller polypeptide carries both glutamine amidotransferase activity and indole-3-glycerolphosphate synthase activity. Various steady-state kinetic parameters as well as the amino acid composition of the two polypeptides were determined.

Amino Acids↗
PubMed1984

The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli.

The alpha subunit is bound with negative cooperativity to the holo beta 2 subunit of tryptophan synthase in phosphate buffer. Thus it is feasible to measure separately the rates of formation both of the stable alpha beta 2 subcomplex from beta 2, and of the mature alpha 2 beta 2 complex from alpha beta 2, using stopped-flow techniques. Addition of each alpha subunit proceeds in two steps; an initial alpha beta protomer is formed rapidly, which subsequently isomerizes slowly to the equilibrium state. The rates of dissociation of both the alpha beta 2 and alpha 2 beta 2 complexes were measured by trapping released alpha subunit with enzymically inactive reduced beta 2 subunit. The reversal of the slow isomerization both determines the rate of dissociation, and accounts for the high overall affinity of the beta protomer for the alpha subunit. The data fit to a sequential assembly mechanism consisting of seven protein species and yields values for most of the rate constants and all of the microscopic equilibrium constants. Negative cooperativity arises from a weaker initial binding of the second alpha subunit, as expressed by its larger off-constant, possibly due to steric hindrance. The kinetics of binding of L-serine and indolepropanol phosphate during the assembly process shows that the beta protomer is already partially activated in the initial alpha beta complex. Full activation is achieved in the slow isomerization reaction. In contrast, the alpha subunit gains high affinity for indolepropanol phosphate only in the isomerization reaction. These observations indicate that the isomerization involves synchronous conformation changes of both alpha and beta protomers.

Bacterial Proteins↗
PubMed1983

The mechanism of binding of L-serine to tryptophan synthase from Escherichia coli.

The mechanism of binding of L-serine to tryptophan synthase, which is the initial phase of the catalytic mechanism, has been studied by steady-state and stopped-flow kinetic techniques. The dependence of three separable rate processes on the concentration of L-serine is compatible with four different enzyme-substrate complexes, one of which lies on a branch in the pathway. By use of L-serine deuterated at the alpha carbon, it is possible to assign the deprotonation of the external aldimine of L-serine with pyridoxal 5'-phosphate to the most rapid observable binding step. Measurements at two pH values show that the rate-determining step in the synthesis of L-tryptophan changes from release of L-tryptophan at the optimal pH of 7.6 to the binding of L-serine at pH 6.5. Measurements at pH 7.6 in the presence of the substrate analogue indolepropanol phosphate show that the stronger binding of L-serine is probably due to stabilization of the catalytically competent enzyme--L-serine complex. At pH 7.6 L-serine is bound far more slowly to the beta 2 subunit than to the alpha 2 beta 2 complex of tryptophan synthase and retains its alpha carbon proton. For the beta 2 subunit, the rate-determining step of tryptophan synthesis is deprotonation of bound L-serine. The effect of bound alpha subunit is to increase both the rate of deprotonation and beta-elimination, shifting the rate-limiting step to the release of L-tryptophan.

Bacterial Proteins↗
PubMed1983

The catalytic mechanism of tryptophan synthase from Escherichia coli. Kinetics of the reaction of indole with the enzyme--L-serine complexes.

The mechanism by which indole condenses with L-serine in the active site of tryptophan synthase was studied by the stopped-flow technique. The single turnover occurs by rapid binding of indole to the pre-formed enzyme--L-serine complex, followed by C--C bond formation, reprotonation of the alpha carbon carbanion of L-tryptophan, and its final release. The effects of isotopic substitution at C-3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L-serine and L-tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme--L-serine complexes, leading to a branched pathway for the central condensation process. The rates of dehydration of L-serine and reprotonation of the carbanion of L-tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C-4' atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the alpha subunit on the reaction catalyzed by the beta 2 subunit.

Catalysis↗
PubMed1983

Effectiveness of the Humphrey Vision Analyzer low vision slide on the partially sighted.

Subjective refractions were performed on a variety of low vision patients using the Humphrey Vision Analyzer (HVA) which incorporates a low vision slide. Twenty-one eyes were examined and results were compared with standardized trial frame refraction findings. The results indicate that the HVA is an effective alternative procedure for some low vision patients, although several factors, including design of the slide, patient alignment, and target illumination, need to be carefully considered if this technique is to replace the conventional low vision trial frame examination.

Adolescent↗
PubMed1982

Small-angle X-ray scattering studies of tryptophan synthase from Escherichia coli and its alpha and beta 2 subunits.

The alpha and beta 2 subunits of tryptophan synthase were investigated by small-angle X-ray scattering. The molecular parameters are: radius of gyration, alpha: 1.95 nm, beta 2: 3.01 nm; maximum particle diameter, alpha: 5.8 nm, beta 2: 10.5 nm; and hydrated volume, alpha: 60 nm3, beta 2 160 nm3. The shape of the alpha subunit can best be described by a circular cylinder, slightly tapered at one end. An elongated elliptical cylinder with its cross section larger in the middle than at the ends was found to be a model equivalent in scattering to the beta 2 subunits. The alpha 2 beta 2 enzyme complex was found to have a radius of gyration of 4.01 nm, a maximum length of 13.5 nm, and a hydrated volume of 270 nm3. No satisfactory fit of the scattering data was obtainable by mere apposition of the models of the alpha and beta 2 subunits. Two cylinders overlapping laterally fit the experimental data considerably better, suggesting changes in the conformation of the subunits on forming the alpha 2 beta 2 complex.

Bacterial Proteins↗
PubMed1981

The mechanism of tryptophan binding to tryptophan synthase from Escherichia coli.

The kinetics of the binding of L-tryptophan to the alpha 2 holo beta 2 complex of tryptophan synthase from Escherichia coli have been measured by rapid-mixing techniques under conditions where tryptophan release is mainly rate-determining in tryptophan synthesis. The dependence of the three observable rate processes on the concentration of L-tryptophan suggests a mechanism in which a rapid binding step is followed by two isomerizations. The effect of the substrate analogue indolepropanol phosphate on the kinetics of binding and synthesis from L-serine and indole supports a branched mechanism with an unproductive enzyme-ligand complex being the major species. The productive enzyme-ligand complex absorbs light at 473 nm but not at 500 nm. These observations, and binding studies with D-tryptophan, suggest that at least two alterative modes of binding of L-tryptophan exist on the enzyme. The effects of protons, indole and indolepropanol phosphate on the three rate processes explain the dependence of kcat on the three non-competitive ligands.

Benzimidazoles↗
PubMed1980

Subunit interactions of tryptophan synthase from Escherichia coli as revealed by binding studies with pyridoxal phosphate analogues.

An improved purification procedure for the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been developed. It consists of DEAE-Sephacel chromatography, followed by hydrophobic chromatography on Sepharose CL 4B, and leads to material with a higher specific activity than reported previously. Inhibition studies, equilibrium dialysis, and spectrophotometric titration were used to study the binding both of pyridoxal phosphate analogues and of bisubstrate analogues. Pyridoxine 5'-phospate and N-phosphopyridoxyl-L-serine bind to the enzyme, but pyridoxamine 5'-phoshate and N-phosphopridoxyl-L-alanine do not. N-Phosphopyridoxyl-L-tryptophan is bound only weakly, although L-tyrptophan binds strongly to the alpha 2 holo beta 2 complex. It is likely that either differences is protonation or in geometry are responsible for the low affinity of the bisubstrate analogues in comparison to that of the external aldimines of either L-serine or L-tyrptophan with pyridoxal 5'-phosphate. As previously found with pyridoxal 5'-phosphate, pyridoxine 5'-phosphate, and N-phosphopryidoxyl-L-serine bind noncooperatively to two identical binding sites in the alpha 2 apo beta 2 complex. The same ligands bind with positive cooperatively to two binding sites in the apo beta 2 subunit. Because the analogues mimic the binding behavior of pyridoxal 5'-phosphate to both proteins, the internal aldimine of pyridoxal 5'-phosphate to the lysine amino group contributes only to the strength of that binding. The nickel apo beta 2 subunit, which is produced by limited proteolysis with trypsin, binds pyroxine 5'-phosphate noncooperatively to two identical sites. Therefore, the loop of polypeptide chain connecting the two autonomous domains of folding must be intact for enzyme activity, for the binding of the alpha subunit, and for cooperative binding of pyridoxine 5'-phosphate.

Binding Sites↗
PubMed1980

Kinetics of cooperative ligand binding to the apo beta 2 subunit of tryptophan synthase and its modulation by the alp ha subunit.

The different binding mechanisms of pyridoxine 5'-phosphate and N-phophopridoxyl-L-serine have been investigated by kinetic studies with rapid reaction techniques. Pyridoxine 5'-phosphate binds in a single rapid step to the alpha 2 apo beta 2 complex and in a single slow step to the nicked apo beta 2 subunit that is obtained by limited proteolysis with trypsin. Both pyridoxine 5'-phosphate and N-phosphopyridoxyl-L-serine bind to the apo beta 2 subunit with a comparatively slow binding step, followed by an event slower isomerization reaction. These findings are consistent with nonexclusive concerted mechanism of cooperative binding but cannot be explained by the simple sequential mechanism. A quantitative fit of the rate and equilibrium data to the concerted mechanism generally yielded the pertinent rate and equilibrium constants. In particular, the same value of L0 = [T0]/[R0] = 200 +/- 50 simultaneously satisfies the data obtained with three different ligands. The comparison of the mechanisms of ligand binding to the three states of the apo beta 2 subunit suggests that the alpha 2 apo beta 2 complex is similar to the high-affinity R state and the nicked apo beta 2 subunit is similar to the low-affinity T state of the apo beta 2 subunit. The slow isonerization involved in the cooperative binding of the ligands to the intact apo beta 2 subunit is discussed in terms of local and concerted conformational changes involving the two autonomously folding domains of the beta protomer.

Apoenzymes↗