Limited proteolysis of the .beta.2-dimer of tryptophan synthase yields an enzymatically active derivative that binds .alpha.-subunits

Kaufmann, Michael; Schwarz, Thomas L.; Jaenicke, Rainer; Schnackerz, Klaus D.; Meyer, Helmut E.; Bartholmes, Peter

doi:10.1021/bi00231a010

Cited by 19 publications

(10 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As a control, the β-subunit of E. coli tryptophan synthase as a typical protein composed of distinct domains connected by a hinge region [10] was also proteolyzed. In contrast to β-tryptophan synthase, aaTHEP1 is resistant to proteolytic cleavage by both trypsin and endoproteinase Glu-C (figure 10).…”

Section: Resultsmentioning

confidence: 99%

“…In contrast to β-tryptophan synthase, aaTHEP1 is resistant to proteolytic cleavage by both trypsin and endoproteinase Glu-C (figure 10). Compared to published experiments on the proteolytic cleavage of β-tryptophan synthase [10,11], the experimental conditions were chosen in a way that fragmented β-tryptophan synthase (~30 and ~10 kDa fragments) already were further degraded. Even under these conditions, aaTHEP1 remains stable although it contains 36 (20 × K + 16 × R) possible trypsin and 17 (17 × E) possible endoproteinase Glu-C cleavage sites as predicted from the sequence.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Thermophile-specific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase

2003

Self Cite

View full text Add to dashboard Cite

BackgroundTo identify thermophile-specific proteins, we performed phylogenetic patterns searches of 66 completely sequenced microbial genomes. This analysis revealed a cluster of orthologous groups (COG1618) which contains a protein from every thermophile and no sequence from 52 out of 53 mesophilic genomes. Thus, COG1618 proteins belong to the group of thermophile-specific proteins (THEPs) and therefore we here designate COG1618 proteins as THEP1s. Since no THEP1 had been analyzed biochemically thus far, we characterized the gene product of aq_1292 which is THEP1 from the hyperthermophilic bacterium Aquifex aeolicus (aaTHEP1).ResultsaaTHEP1 was cloned in E. coli, expressed and purified to homogeneity. At a temperature optimum between 70 and 80°C, aaTHEP1 shows enzymatic activity in hydrolyzing ATP to ADP + Pi with kcat = 5 × 10-3 s-1 and Km = 5.5 × 10-6 M. In addition, the enzyme exhibits GTPase activity (kcat = 9 × 10-3 s-1 and Km= 45 × 10-6 M). aaTHEP1 is inhibited competitively by CTP, UTP, dATP, dGTP, dCTP, and dTTP. As shown by gel filtration, aaTHEP1 in its purified state appears as a monomer. The enzyme is resistant to limited proteolysis suggesting that it consists of a single domain. Although THEP1s are annotated as "predicted nucleotide kinases" we could not confirm such an activity experimentally.ConclusionSince aaTHEP1 is the first member of COG1618 that is characterized biochemically and functional information about one member of a COG may be transferred to the entire COG, we conclude that COG1618 proteins are a family of thermophilic NTPases.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Thermophile-specific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase

2003

Self Cite

View full text Add to dashboard Cite

show abstract

“…Limited Proteolysis and Reaction Specificity-Limited proteolysis is an ideal tool to detect solvent-exposed loops lacking defined secondary structure as described for the hinge region in the ␤-subunit of tryptophan synthase from E. coli (106) or for the connector region of tryptophan synthase from S. cerevisiae (86). Using endoproteinase Glu-C (and some other endoproteinases; data not shown), only one major cleavage site could be detected.…”

Section: Discussionmentioning

confidence: 99%

Multifunctional Tryptophan-synthesizing Enzyme

Schwarz

Uthoff

Meyer

et al. 1997

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

After developing a suitable procedure to produce large amounts of Euglena gracilis as well as a reliable protocol to purify the multifunctional tryptophan-synthesizing enzyme derived from it (Schwarz, T., Bartholmes, P., and Kaufmann, M. (1995) Biotechnol. Appl. Biochem. 22, 179 -190), we here describe structural and catalytic properties of the multifunctional tryptophansynthesizing enzyme. The kinetic parameters k cat of all five activities and K m for the main substrates were determined. The relative molecular weight under denaturing conditions as judged by SDS-polyacrylamide gel electrophoresis is 136,000. Cross-linking as well as gel filtration experiments revealed that the enzyme exists as a homodimer. Neither intersubunit disulfide linkages nor glycosylations were detected. On the other hand, the polypeptide chains are blocked N-terminally. Complete tryptic digestion of the protomer, high pressure liquid chromatography separation of the resulting peptides, and N-terminal sequence analysis of homogenous peaks as judged by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry was performed. Depending on the sequenced peptides, alignments to all entries of the SwissProt data base resulted in both strong sequence homologies to known Trp sequences and no similarities at all. Proteolytic digestion under native conditions using endoproteinase Glu-C uncovered one major cleavage site yielding a semistable, Nterminally blocked fragment with a molecular weight of 119,000. In addition, an increase in ␤-elimination accompanied by a decrease in ␤-replacement activity of the ␤-reaction during proteolysis was observed. In most procaryotes, tryptophan synthase is organized as a tetrameric bienzyme complex consisting of a central ␤ 2 -dimer (TRPS␤) combined with two peripheral ␣-subunits (TRPS␣). The Escherichia coli and Salmonella typhimurium enzymes in particular have been intensively investigated in the past (1, 2). In fungi such as Saccharomyces cerevisiae and Neurospora crassa, tryptophan synthase is a dimer of identical bifunctional polypeptides (3,4,5). In contrast, the enzyme isolated from Euglena gracilis, here designated as multifunctional tryptophan-synthesizing enzyme (MTSE), is capable of catalyzing all reactions shown in Scheme I. Although this discovery was made 2 decades ago (6, 7), no thorough further characterization of this multifunctional enzyme has been published so far, probably due to the excessive expenditure to cultivate sufficient amounts of E. gracilis and the difficulties of purifying MTSE with outdated technology. Using state of the art equipment and materials, we established a reliable and simple procedure to obtain MTSE from E. gracilis (8). Here we present both a reinvestigation of earlier initial findings and new data with respect to structural and functional properties of the enzyme. EXPERIMENTAL PROCEDURESChemicals, Enzymes, and E. coli Strains-If not otherwise stated, all chemicals (reagent or ultrapure grade) were obtained from Sigma (Deisenhofen, Germany) or Merck...

show abstract

“…This hinge region is included in the long stretch from 260 -310 (with a loop conformation), which forms one side of the intersubunit tunnel of the ␤ subunit (14). The substitution (28,54) or proteolysis (55)(56)(57) in the "hinge" region of the ␤ subunit reduces the stimulation activity, the substrate affinity, and the association between the ␣ and ␤ subunits. Thus, this region is believed to play a critical role in the conformational change upon the subunit association (1).…”

Section: Roles Of Hydrogen Bonds Between Subunits In Tsase Complexmentioning

confidence: 99%

Roles of Hydrogen Bonding Residues in the Interaction between the α and β Subunits in the Tryptophan Synthase Complex

Hiraga¹

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

The interaction of the ␣ subunit with the ␤ 2 subunit of tryptophan synthase is known to be necessary for the activation of each subunit and for the catalytic efficiency of the ␣ 2 ␤ 2 complex. To elucidate the roles of hydrogen bonds in the interaction site between the ␣ and ␤ subunits for subunit association, eight mutant ␣ subunits at five hydrogen bonding residues (N104D, N104A, N108D, N108A, E134A, E135A, N157D, and N157A) were constructed, and the thermodynamic parameters of association with the ␤ subunit were obtained using a titration calorimeter. The N104D and N104A mutations remarkably decreased the stimulation activities, the association constants, and the association enthalpies. Although the association constant and the stimulation activities of E134A were reduced in the absence of salt, the change in the association enthalpy was relatively small, and the addition of salt could repair its defects. The substitutions at positions 135 and 157 did not affect the stimulation activity and decreased the Gibbs energy of association corresponding to the defect in 1 mol of hydrogen bond. The present results suggest that the ␣ subunit which has a mutation at position 104 cannot fold into an intact conformation upon complex formation, resulting in reduced stimulation activities. The hydrogen bond with Asn-104, which is a conserved residue among 16 microorganisms, was especially important for ␣/␤ interaction and mutual activation.Protein-protein interactions play a central role in many physiologically important reactions. The tryptophan synthase complex is an excellent model enzyme for exploring the molecular recognition mechanism in protein-protein interaction (1, 2). Bacterial tryptophan synthase is an ␣ 2 ␤ 2 complex. The separated ␣ and ␤ subunits catalyze inherent reactions termed the ␣ and ␤ reactions, respectively (3-5). When the ␣ and ␤ subunits combine to form the ␣ 2 ␤ 2 complex, the enzymatic activity of each subunit is stimulated by 1 to 2 orders of magnitude. It has been reported that this activation is due to conformational changes in both subunits upon ␣/␤ subunit interaction (reviewed in Ref. 1).Some intermolecular electrostatic interactions (hydrogen bonds and salt bridges) and hydrophobic (packing) interactions were observed in many strongly bound complexes (6 -8). In particular, electrostatic interaction is thought to play a critical role in protein-protein interaction (8 -11). We focused on hydrogen bonds in the interaction site between the ␣ and ␤ subunits of tryptophan synthase, termed herein "␣/␤ subunit hydrogen bonds." A thermodynamic study of the association of proteins having mutations at interface residues is important to elucidate the mechanisms of the subunit interaction in the tryptophan synthase complex. Also, it should provide useful information for understanding the energetic contribution of the hydrogen bonds that stabilize the protein complex. The amino acid sequences of the ␣ and ␤ subunits from Escherichia coli are highly similar to those from Salmonella typhimurium (12); the...

show abstract

Limited proteolysis of the .beta.2-dimer of tryptophan synthase yields an enzymatically active derivative that binds .alpha.-subunits

Cited by 19 publications

References 30 publications

Thermophile-specific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase

Thermophile-specific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase

Multifunctional Tryptophan-synthesizing Enzyme

Roles of Hydrogen Bonding Residues in the Interaction between the α and β Subunits in the Tryptophan Synthase Complex

Contact Info

Product

Resources

About