Direct identification of the primary nucleophile of thioredoxin f.

Thioredoxin, by virtue of the proximal active-site sulfhydryls (Trp-Cys-Gly-Pro-Cys), catalyzes thiol-disulfide exchange with specific target enzymes. Considerable data (chemical modification, spectroscopic, and crystallographic) have implicated the cysteinyl residue nearest the N terminus of thioredoxin as the primary nucleophile; however, direct proof has been lacking. Proof is now provided by characterization of site-directed mutants of thioredoxin f with respect to activation of chloroplastic fructose-1,6-bisphosphatase (FBPase). The C49S mutant retains the capacity to activate FBPase, whereas the C46S mutant is totally lacking in this regard. Based on kinetics of FBPase activation, wild-type and C49S thioredoxins exhibit half-saturation values of 0.9 and 4 microM, respectively. Lack of activation by C46S is not because of failure to interact with FBPase, for it exhibits a Ki of 5 microM in competition with wild-type thioredoxin. Therefore, in the normal thioredoxin-catalyzed reduction pathway, Cys-46 is the nucleophile required to attack the disulfide of the substrate and Cys-49 serves to cleave the mixed disulfide intermediate, thus allowing for the release of oxidized thioredoxin and the reduced target enzyme.

Conformational flexibility of the regulatory site of phosphoribulokinase as demonstrated with bifunctional reagents.

Phosphoribulokinase (PRK) is one of several chloroplastic enzymes whose activity is regulated by thiol-disulfide exchange via thioredoxin. Activation entails reduction of an active-site disulfide bond between Cys16 and Cys55. Bifunctional cross-linking reagents have been used to approximate the interresidue distance between Cys16 and Cys55, an issue which impinges on the relative conformational states of the activated and deactivated forms of the enzyme. Spinach PRK is rapidly inactivated by stoichiometric levels of 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (FNPS) or 1,5-difluoro-2,4-dinitrobenzene (DFNB), which span 9 and 3.5 A, respectively. ATP, but not ribulose 5-phosphate, retards the rate of inactivation, suggesting that modification has occurred at the nucleotide binding domain of the active site. Sulfhydryl modification is indicated by partial reversibility of inactivation as effected by exogenous thiols. Tryptic mapping by reverse-phase chromatography of [14C]carboxymethylated enzyme, subsequent to its reaction with either FNPS or DFNB, demonstrates modification of Cys16 and Cys55 by both reagents, and formation of only one major chromophoric peptide in each case. On the basis of the sequence analysis of the purified chromophoric peptides, Cys16 and Cys55 are cross-linked by both FNPS and DFNB. Thus, the intrasubunit distance between the beta-sulfhydryls of Cys16 and Cys55 is dynamic rather than static. Diminished conformational flexibility upon oxidation of the regulatory sulfhydryls to a disulfide may be partially responsible for the concomitant loss of enzymatic activity.

Analysis of the tumor-associated antigen TSP-180. Identity with alpha 6-beta 4 in the integrin superfamily.

The tumor-associated antigen complex, TSP-180, was previously defined in carcinoma cell lines and found to be expressed in higher amounts in tumor than in normal tissue. Here, the mouse TSP-180 complex is shown to consist of three related proteins (bands 1, 2, and 3) associated with a distinct protein (band 5) that is probably derived from a precursor protein (band 4). All of these proteins are cell surface glycoproteins, and the largest protein (band 1) can be readily labeled with 32PO4. The mouse TSP-180 complex described here strongly resembles the recently described human integrin alpha 6-beta 4 complex. This homology was confirmed using two distinct rat anti-alpha 6 monoclonal antibodies, each of which recognized both human alpha 6-beta 4 and mouse TSP-180 complexes. Furthermore, the TSP-180 band 5 protein (mouse alpha 6) had an N-terminal sequence identical to that of human alpha 6. Finally, two different monoclonal antibodies are described, 346-11A and 439-9B, which directly recognize the multiple forms of mouse and human beta 4 proteins, respectively.

Cloning of authentic human epidermal growth factor as a bacterial secretory protein and its initial structure-function analysis by site-directed mutagenesis.

A synthetic chimeric gene, coding for the human epidermal growth factor fused to the signal peptide of Escherichia coli alkaline phosphatase, was cloned into E. coli under the transcriptional control of the trp-lac (tac) promoter. Following induction with isopropylthiogalactoside, the secretion of the correctly processed protein product into the bacterial periplasm was detected and quantitated by its specific binding to the epidermal growth factor receptor. The purified protein was identical to authentic human epidermal growth factor in size, amino acid composition, primary sequence, receptor binding, and stimulation of receptor protein-tyrosine kinase activity. Based on interspecies homologies, structural considerations, and reported studies with peptide fragments, structure-function analysis was initiated with alterations of targeted amino acid residues by oligonucleotide-directed mutagenesis. The receptor binding affinity of each mutant, relative to the wild type, was measured by both radioreceptor competition and receptor tyrosine kinase stimulation assays. In general, the values obtained by the two methods were in agreement for each species of epidermal growth factor and followed the order: wild type greater than Glu24----Gly greater than Asp27----Gly much greater than Pro7----Thr greater than Tyr29----Gly greater than Leu47----His. The relatively low values obtained with the last two mutants suggest that Tyr29 and Leu47 may be important for the biological activity of human epidermal growth factor.

Thiol-sensitive promoters of Escherichia coli.

Mu dX(lac) insertion mutants of Escherichia coli CSH50 in which the expression of the lacZ gene was sensitive to the presence of exogenous 1-thioglycerol or dithiothreitol were isolated. Both stimulatory and inhibitory mutants were found. The existence of several thiol-sensitive promoters suggests that exogenous thiols may provoke global stress responses in E. coli.

Characterization of the regulatory thioredoxin site of phosphoribulokinase.

Phosphoribulokinase is light-regulated via thioredoxin by reversible oxidation/reduction of sulfhydryl/disulfide groups. To identify the cysteinyl residues that are involved in regulation, the S-carboxymethyl labeling patterns of the fully reduced (active) and oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced, [14C]carboxymethylated enzyme contained four labeled peptides, all of which were purified and sequenced by Edman degradation. If the enzyme was oxidized by 5,5'-dithiobis-(2-nitrobenzoic acid) prior to carboxymethylation and tryptic digestion, only two labeled peptides were observed, thereby revealing the identity of the regulatory cysteines as Cys-16 and Cys-55. The former was previously implicated as part of the nucleotide-binding domain of the active site (Porter, M.A., and Hartman, F.C. (1986) Biochemistry 25, 7314-7318), a conclusion reinforced by the present observation that the sequence around the Cys-16 is similar to a consensus sequence of ATP-binding sites from a number of proteins of diverse phylogenetic origin (Higgins, C.F., Hiles, I.D., Salmond, G.P.C., Gill, D.R., Downie, J.A., Evans, I.J., Holland, I.B., Gray, L., Buckel, S.D., Bell, A.W., and Hermondson, M. (1986) Nature 323, 448-450). The regulatory disulfide of phosphoribulokinase was found to be intrasubunit based on the stoichiometry of the oxidation and the failure to resolve oxidized and reduced enzyme by gel filtration under dissociation conditions.

Distance between two active-site lysines of ribulose bisphosphate carboxylase from Rhodospirillum rubrum.

In the absence of a three-dimensional structure of ribulose-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase(dimerizing), EC 4.1.1.39], we have probed the distance between two active-site lysyl residues (Lys-166 and Lys-329) of the Rhodospirillum rubrum enzyme with 4,4'-diisothiocyano-2,2'-disulfonate stilbene, a covalent cross-linking reagent that spans 12 A. The reagent rapidly inactivated the carboxylase, and a competitive inhibitor provided substantial protection. To remove products arising from intersubunit or intermolecular cross-linking, the inactivated enzyme was subjected to gel filtration in the presence of urea. Inspection of a tryptic digest of the isolated monomeric fraction revealed that more than half of the incorporated reagent was associated with a single peptide. This peptide was purified by gel filtration, followed by high HPLC. Compositional and sequence analyses of the purified peptide established that it was composed of two chains, encompassing positions 149-168 and 314-337 of the original protein subunit and connected by a cross-link between Lys-166 and Lys-329. Thus, the two active-site lysines of the carboxylase can be juxtaposed within 12 A, a finding that is consistent with their purported proximity to ribulose bisphosphate in the enzyme-substrate complex. The cross-link was not formed when the carboxylase was treated with the reagent either in the presence of a transition-state analogue (carboxyarabinitol bisphosphate) or in the absence of CO(2) and Mg(2+), conditions under which the enzyme exists in a deactivated form.

Purification and characterization of ribulose-5-phosphate kinase from spinach.

An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation.

Characterization of an active-site peptide modified by glyoxylate and pyridoxal phosphate from spinach ribulosebisphosphate carboxylase/oxygenase.

Activated ribulosebisphosphate carboxylase/oxygenase from spinach was treated with glyoxylate plus or minus the transition-state analog, carboxyarabinitol bisphosphate, or the inactive enzyme with pyridoxal phosphate plus or minus the substrate, ribulose bisphosphate. Covalently modified adducts with glyoxylate or pyridoxal phosphate were formed following reduction with sodium borohydride. The derivatized enzymes were carboxymethylated and digested with trypsin; the labeled peptides which were unique to the unprotected samples were purified by ion-exchange chromatography and gel filtration. Both glyoxylate and pyridoxal phosphate were associated with only one major peptide, which in each case was subjected to amino acid analysis and sequencing. The sequence was -Tyr-Gly-Arg-Pro-Leu-Leu-Gly-Cys(Cm)-Thr-Ile-Lys-Lys*-Pro-Lys-, with both reagents exhibiting specificity for the same lysine residue as indicated by the asterisk. This peptide is identical to that previously isolated from spinach carboxylase labeled with either of two different phosphorylated affinity reagents and homologous to one from Rhodospirillum rubrum carboxylase modified by pyridoxal phosphate. The species invariance of this lysine residue, number 175, and the substantial conservation of adjacent sequence support the probability for a functional role in catalysis of the lysyl epsilon-amino group.

Complete primary structure of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

Of the 14 cyanogen bromide fragments derived from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, four are too large to permit complete sequencing by direct means [F. C. Hartman, C. D. Stringer, J. Omnaas, M. I. Donnelly, and B. Fraij (1982) Arch. Biochem. Biophys. 219, 422-437]. These have now been digested with proteases, and the resultant peptides have been purified and sequenced, thereby providing the complete sequences of the original fragments. With the determination of these sequences, the total primary structure of the enzyme is provided. The polypeptide chain consists of 466 residues, 144 (31%) of which are identical to those at corresponding positions of the large subunit of spinach ribulosebisphosphate carboxylase/oxygenase. Despite the low overall homology, striking homology between the two species of enzyme is observed in those regions previously implicated at the catalytic and activator sites.

Isolation, characterization, and crystallization of ribulosebisphosphate carboxylase from autotrophically grown Rhodospirillum rubrum.

Serial culture of Rhodospirillum rubrum with 2% CO2 in H2 as the exclusive carbon source resulted in a rather large fraction of the soluble protein (greater than 40%) being comprised of ribulosebisphosphate carboxylase (about sixfold higher than the highest value previously reported). Isolation of the enzyme from these cells revealed that it has physical and kinetic properties similar to those previously described for the enzyme derived from cells grown on butyrate. Notably, the small subunit (which is a constituent of the carboxylase from eucaryotes and most procaryotes) was absent in the enzyme from autotrophically grown R. rubrum. Edman degradation of the purified enzyme revealed that the NH2 terminus is free (in contrast to the catalytic subunit of the carboxylase from eucaryotes) and that the NH2-terminal sequence is Met-Asp-Gln-Ser-Ser-Arg-Tyr-Val-Asn-Leu-Ala-Leu-Lys-Glu-Glu-Asp-Leu-Ile-Ala-Gly-Gly-Glx-His-Val-Leu-. Crystals of the enzyme were readily obtained by dialysis against distilled water.

Inactivation of ribulosebisphosphate carboxylase by modification of arginyl residues with phenylglyoxal.

Phenylglyoxal rapidly and completely inactivates spinach and Rhodospirillum rubrum ribulosebisphosphate carboxylases. Inactivation exhibits pseudo-first-order kinetics and a reaction order of approximately one for both enzymes, suggesting that modification of a single residue per protomeric unit suffices for inactivation. Loss of enzymic activity is directly proportional to incorporation of [14C]phenylglyoxal until only 30% of the initial activity remains. For both enzymes, extrapolation of incorporation to 100% inactivation yields 4-5 mol of [14C]phenylglyoxal per mol protomer. Amino acid analyses confirm the expected 2:1 stoichiometry between phenylglyoxal incorporation and arginyl modification and suggest that other kinds of amino acid residues are not modified. (Thus, inactivation correlates with modification of 2-3 arginyl residues per protomer). The substrate ribulose bis-phosphate and some competitive inhibitors reduce the rates of inactivation of the carboxylases and prevent modification of about 0.5-1.0 arginyl residue per protomer. Inactivation is therefore a consequence of modification of a small number of residues out of the 35 and 29 total arginyl residues per protomer in spinach and R. rubrum carboxylases, respectively.

Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate carboxylase/oxygenase modified by the affinity label N-bromoacetylethanolamine phosphate.

We reported earlier (Schloss, J. V., and Hartman, F. C. (1977) Biochem. Biophys. Res. Commun. 77, 230-236) that N-bromoacetylethanolamine phosphate is an affinity label for spinach ribulosebisphosphate carboxylase/oxygenase. We now show inactivation to be correlated directly with the alkylation either of a single lysyl residue (in the presence of Mg2+) or of 2 different cysteinyl residues (in the absence of Mg2+), consistent with the likelihood that these residues are located in the active site region. This proposition is further supported by the demonstration that the residues are protected from alkylation by substrate, a competitive inhibitor, or the transition state analog 2-carboxyribitol bisphosphate. Tryptic peptides that contain the modified residues have been isolated and sequenced. One of the 2 cysteinyl residues that are subject to alkylation is only 3 residues distant in sequence from the lysyl residue modified by bromoacetylethanolamine phosphate. This lysyl residue is identical with 1 of the 2 lysyl residues alkylated by the previously described affinity label, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate (Stringer, C. D., and Hartman, F. C. (1978) Biochem. Biophys, Res. Commun. 80, 1043-1048).