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K Kirschner

Publications and source records attributed to K Kirschner.

15 recordsLinked to original sources
PubMed1992

Collapsed intermediates in the reconstitution of dimeric aspartate aminotransferase from Escherichia coli.

Aspartate aminotransferase from Escherichia coli, which had been denatured by guanidinium chloride, refolded and reassembled to active dimers in two distinct phases. The unfolded monomer U collapsed within 20 s to an intermediate I* that was inactive, fluoresced more strongly than, but had the same peptide CD signal as the native dimer. The formation of crosslinkable dimers, as well as the recovery of enzyme activity, occurred with a biphasic progress curve which was independent of protein concentration. The half-lives of the two phases were 100 s and 2000 s. The data are consistent with a three-step mechanism, in which the overall rate of reassembly is determined by an isomerization of I* to the assembly-competent monomer M. The latter does not accumulate because it dimerizes rapidly to the active enzyme (D). Reassembly of the enzyme from the compact intermediate M*, which is stable at 1.0 M guanidinium chloride, also proceeded in a rapid and a slow phase. Moreover, the formation of M* from the unfolded state was rapid, whereas its refolding to the native dimer was slow. Both the transient intermediate I* and the equilibrium intermediate M* qualify as 'collapsed intermediate' or 'molten globule' states.

Aspartate Aminotransferases↗
PubMed1992

Stable substructures of eightfold beta alpha-barrel proteins: fragment complementation of phosphoribosylanthranilate isomerase.

The (beta alpha)8 (or "TIM")-barrel protein phosphoribosylanthranilate isomerase from Saccharomyces cerevisiae was cleaved between the sixth and seventh beta alpha module to test the capacity of the resulting fragments to adopt native format autonomously. The fragments, which were expressed from separate coding sequences, were soluble and monomeric. The amino-terminal fragment p1 was compact, possessed an almost nativelike far-UV but a strongly reduced near-UV CD spectrum, and unfolded cooperativity with guanidinium chloride. In contrast, the carboxyl-terminal fragment p2 was less compact than fragment p1, possessed only a weak far-UV and no detectable near-UV CD spectrum, and unfolded noncooperatively. The fragments assembled stoichiometrically to a complex with Kd = 0.2 microM, which was enzymically almost fully active. The rate of assembly was limited by a first-order process, probably the isomerization of the carboxyl-terminal fragment p2 to an assembly-competent structure. These results support a folding mechanism that comprises an intermediate with the first six beta alpha units folded in roughly native format and the last two beta alpha units partially unfolded. The similar behavior of the analogous fragments of the alpha subunit of tryptophan synthease supports the hypothesis that these two (beta alpha)8-barrel proteins have evolved from a common ancestor.

Aldose-Ketose Isomerases↗
PubMed1992

A fully active variant of dihydrofolate reductase with a circularly permuted sequence.

The amino acid sequence of mouse dihydrofolate reductase was permuted circularly at the level of the gene. By transposing the 3'-terminal half of the coding sequence to its 5' terminus, the naturally adjacent amino and carboxyl termini of the native protein were fused, and one of the flexible peptide loops at the protein surface was cleaved. The steady-state kinetic constants, the dissociation constants of folate analogues, and the degree of activation by both mercurials and salt as well as the resistance toward digestion by trypsin were almost indistinguishable from those of a recombinant wild-type protein. Judged by these criteria, the circularly permuted variant has the same active site and overall structure as the wild-type enzyme. The only significant difference was the lower stability toward guanidinium chloride and the lower solubility of the circularly permuted variant. This behavior may be due to moving a mononucleotide binding fold from the interior of the sequence to the carboxyl terminus. Thus, dihydrofolate reductase requires neither the natural termini nor the cleaved loop for stability, for the conformational changes that accompany catalysis as well as the binding of inhibitors, and for the folding process.

Amino Acid Sequence↗
PubMed1992

The importance of surface loops for stabilizing an eightfold beta alpha barrel protein.

An important step in understanding how a protein folds is to determine those regions of the sequence that are critical to both its stability and its folding pathway. We chose phosphoribosyl anthranilate isomerase from Escherichia coli, which is a monomeric representative of the (beta alpha)8 barrel family of proteins, to construct a variant that carries an internal tandem duplication of the fifth beta alpha module. This (beta alpha)9 variant was enzymically active and therefore must have a wild-type (beta alpha)8 core. It had a choice a priori to fold to three different folding frames, which are distinguished by carrying the duplicated segment as an insert into one out of three different loops. Steady-state kinetic constants, the fluorescence properties of a crucial tryptophan residue, and limited proteolysis showed that the stable (beta alpha)9 variant carries the insertion between beta-strand 5 and alpha-helix 5. This preference can be explained by the important role of loops between alpha helices and beta strands in stabilizing the structure of the enzyme.

Amino Acid Sequence↗
PubMed1979

N-(5-Phosphoribosyl)anthranilate isomerase-indoleglycerol-phosphate synthase. 2. Fast-reaction studies show that a fluorescent substrate analogue binds independently to two different sites.

The mechanism of binding of reduced 1-(2-carboxyphenylamino)-1-deoxyribulose 5-phosphate (rCdRP) to two different binding sites on the bifunctional enzyme is determined by kinetic studies, using temperature-jump and stopped-flow equipment with fluorescence detection. Two rapid binding processes and a comparatively slow isomerization process are observed over a wide range of enzyme and rCdRP concentrations. Kinetic measurements with low concentrations of rCdRP show that the isomerization is coupled only to the more rapid of the two binding reactions that involves the active site of indoleglycerol-phosphate synthase. The slower of the two binding reactions represents rCdRP binding in one step to the active site of (phosphoribosyl)anthranilate isomerase. The simplest mechanism explaining quantitatively the dependence of the relaxation times on concentration consists of rCdRP binding to two sites on the enzyme that are intrinsically different and independent, even to the extent that a ligand-induced isomerization of one site is not transmitted to the other site. Simulation studies show that the concentration dependences of the amplitudes of the three relaxation processes are also consistent with the mechanism. The results are discussed in terms of two autonomous domains of folding of the polypeptide chain.

Carboxy-Lyases↗
PubMed1979

Affinity chromatography of tryptophan synthase from Escherichia coli. Systematic studies with immobilized tryptophanol phosphate.

Inhibition studies and affinity chromatography indicate that derivatives of tryptophanol phosphate are suitable ligands for the affinity chromatography of tryptophan synthase. A phenyl group on the spacer arm strengthens the interaction of immobilized tryptophanol phosphate with the enzyme. The alpha 2 beta 2 complex specifically requires the presence of 0.3--0.5 M phosphate ions for binding. The alpha subunit binds in dilute Tris buffer, but its binding is also enhanced by the presence of phosphate ions. The beta 2 subunit binds unspecifically but strongly to the affinity material and to a variety of other immobilized hydrophobic ligands. Binding studies with suspensions of affinity material show that the alpha subunit interacts rapidly and reversibly. Indoleglycerol phosphate and indolepropanol phosphate release bound alpha 2 beta 2 complex and alpha subunit in a competitive manner, indicating that the interaction occurs biospecifically, i.e. via the active site of alpha subunit. L-Serine is a non-competitive inhibitor of binding. These results are discussed with regard to the composite-active-site hypothesis [T. E. Creighton (1970) Eur. J. Biochem, 13, 1--10]. Both the alpha subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli can be obtained with high yields and in homogenous form by absorption to the affinity material from partially purified preparations. Elution is achieved with linear gradients either of indolepropanol phosphate or of indoleglycerol phosphate or, in the case of the complex, of L-serine. At the low concentrations of the complex found in crude extracts of wild-type E. coli cells, the unexpectedly high affinity of the beta 2 subunit for hydrophobic ligands leads to partial dissociation of the complex.

Chromatography, Affinity↗
PubMed1977

[Assembly and function of organized enzymes (author's transl)].

When different enzymes form complexes with one another they gain new capabilities of function. Tryptophan synthase is discussed as an illustrative example in which formation of the complex leads to mutual activation of the component subunits. Kinetic studies show that conformational changes play an important role in this process. As the extreme case of self-organisation, structural genes of different enzymes might have been fused in the course of evolution, giving rise to multifunctional enzymes.

Biological Evolution↗
PubMed1976

Cooperative and noncooperative binding of pyridoxal 5'-phosphate to tryptophan synthase from Escherichia coli.

An improved purification procedure for the beta2 subunit of tryptophan synthase from from Escherichia coli has led to an essentially pure and stable preparation with a specific enzymatic activity that is 30% higher than the previously reported maximum value. Sedimentation analysis shows that the apo-beta2 subunit is monodisperse and dimeric down to a concentration of 0.02 mg of protein/ml. The binding of pyridoxal 5'-phosphate (pyridoxal-P) to the apo-beta2 subunit and to the alpha2-apo-beta2 complex was studied by equilibrium dialysis and spectroscopic titration. Both the beta2 subunit and the alpha2beta2 complex bind 2 mol of pyridoxal-P with no unspecific binding observable at higher concentrations of pyridoxal-P. The binding of pyridoxal-P to the apo-beta2 subunit is cooperative (Hill coefficient nH = 1.7). The data have been fitted to the Adair equation, yielding the apparent microscopic dissociation constants for the complexes with one and two bound ligand molecules. They differ by a factor of 38, suggesting that the apo- and holo-beta2 subunits have distinct conformations. The binding of pyridoxal-P to the alpha2-apo-beta2 complex is noncooperative with a value of the dissociation constant intermediate between the two values of the beta2 subunit. This finding suggests that the alpha subunit may stabilize a third conformational state of the beta2 subunit.

Apoproteins↗
PubMed1976

The mechanism of the synthesis of indoleglycerol phosphate catalyzed by tryptophan synthase from Escherichia coli. Steady-state kinetic studies.

The mechanism of indoleglycerol phosphate synthesis from indole and D-glyceraldehyde 3-phosphate catalyzed by tryptophan synthase has been investigated by steady-state kinetic techniques. The equilibrium constant and the progress curves were measured by use of the difference in absorbance between indole and indoleglycerol phosphate. Stopped-flow measurements show that only the non-hydrated form of D-glyceraldehyde 3-phosphate serves as substrate. The product analogue indolepropanol phosphate was used as an inhibitor to discriminate between possible mechanisms. The data agree well with an ordered addition mechanism with D-glyceraldehyde 3-phosphate adding first. Mechanisms involving random addition of substrates or ordered addition with indole adding first can be excluded because indolepropanol phosphate is a competitive inhibitor only towards glyceraldehyde 3-phosphate. The high affinity of tryptophan synthase for indoleglycerol phosphate leads to product inhibition even at small extents of reaction. Glyceraldehyde 3-phosphate combines with the enzyme with an apparent second-order rate constant, which is not diffusion controlled and generates a site with high affinity for indole.

Chemical Phenomena↗
PubMed1976

Steady-state kinetic studies of the synthesis of indoleglycerol phosphate catalyzed by the alpha subunit of tryptophan synthase from Escherichia coli. Comparison with the alpha2 beta2-complex.

For the alpha subunit of tryptophan synthase and at constant concentration of D-glyceraldehyde 3-phosphate the saturation curves with respect to indole concentration are weakly sigmoidal. This phenomenon can be explained by interaction between indole bound to the effector site established previously and the active center of the monomeric alpha subunit. Kinetic studies of the inhibition of indoleglycerol phosphate synthesis by the analogue indolepropanol phosphate show that the inhibition is competitive with respect to D-glyceraldehyde 3-phosphate and non-competitive with respect to indole. Mechanisms with random addition of substrates or ordered addition with indole binding first can therefore be excluded. A quantitative fit of the data has been obtained to an ordered addition mechanism with D-glyceraldehyde 3-phosphate binding first and with a distribution of the enzyme between two states differing in V, governed by the binding of indole to the effector site. The kinetic constants obtained for the alpha subunit have been compared with those of the alpha 2 beta 2 complex of tryptophan synthase. Protein-protein interaction of the alpha subunit with the beta 2 subunit (a) does not alter the catalytic of the indoleglycerol phosphate synthesis, (b) suppresses the substrate activation by indole, and (c) changes the various equilibrium, rate and steady-state constants in the sense of conveying higher substrate specificity and catalytic efficiency to the alpha-subunit. The occurrence of local and gross conformational changes in the tryptophan synthase system is discussed.

Binding Sites↗
PubMed1976

The binding of indole to the alpha-subunit and beta2-subunit and to the alpha2beta2-complex of tryptophan synthase from Escherichia coli. Identification of a second indole-binding site on the alpha-subunit.

The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual alpha and beta2-subunits and to the alpha2beta2-complex of tryptophan synthase was studied by equilibrium dialysis. The use of [14C]indole and indolepropanol [32P]phosphate permitted simultaneous binding studies to be carried out. Competition between indole and indolepropanol phosphate in binding to a particular site was taken as evidence for that site being part of the active site of the alpha-subunit. The binding of indole to the active site of the alpha-subunit is weak (Kd = 18mM). A second distinct site binds indole more strongly (Kd = 1.5 mM) and interacts with the active site indirectly. It is therefore designated an effector site. Furthermore, the binding of indole and/or indolepropanol phosphate appears to stabilize different conformations of the alpha-subunit. The beta2-subunit binds indole only weakly (Kd = 12 mM) to many (n = 10) sites per polypeptide chain. The alpha2beta2-complex retains one or two sites per alphabeta-equivalent of relatively high affinity (Kd = 1.2 mM). The active sites of the component alpha and beta-subunits probably belong to the second class of many (n = 40) sites of low (Kd = 30 mM) affinity for indole. These findings support conclusions from the literature that both bi-substrate reactions involving indole catalyzed by tryptophan synthase and its subunits must follow strictly ordered addition mechanisms with the respective other substrate adding first.

Binding Sites↗
PubMed1975

The tryptophan synthase from Escherichia coli. An improved purification procedure for the alpha-subunit and binding studies with substrate analogues.

An improved method is described for the purification of the alpha-subunit of tryptophan synthase from Escherichia coli. The standard manganese chloride and acid-precipitation steps have been replaced by rapid and efficient chromatographic procedures. Indoleethanol phosphate, indoleprapanol phosphate and indolebutanol phosphate have been synthesized. They are not cleaved by tryptophan synthase and are strictly competitive inhibitors versus indoleglycerol phosphate. The inhibition constant decreases as the number of methylene groups in the side chain increases. This may reflect an improved accommodation of the indole and phosphate moienerated by binding indole, indoleglycerol phosphate and indolepropanol phosphate to the alpha-subunit are very similar. This reflects the transfer of the indole moiety to an hydrophobic environment within the active center. The binding of indolepropanol phosphate to the alpha2beta2-complex perturbs the spectrum of pyridoxal 5'-phosphate located in the beta2-subunit. This demonstrates direct or indirect interactions between the component active sites. Bind studies by spectrophotometric titration and equilibrium dialysis with indolepropanol [32P]phosphate show that there is only one binding site per equivalent of alpha-subunit. Complex formation with the beta2-subunit increases the affinity of the alpha-subunit for indolepropanol phosphate, It is a general consequence of protein-protein interaction in this system.

Binding Sites↗