8-(4-Bromo-2,3-dioxobutylthio)guanosine 5'-triphosphate: a new affinity label for purine nucleotide sites in proteins.

A new affinity label, 8-(4-bromo-2,3-dioxobutylthio)guanosine 5'-triphosphate (8-BDB-TGTP), has been synthesized by initial reaction of GTP to form 8-Br-GTP, followed by its conversion to 8-thio-GTP, and finally coupling with 1,4-dibromobutanedione to produce 8-BDB-TGTP. 8-BDB-TGTP and its synthetic intermediates were characterized by thin-layer chromatography, UV, (31)P NMR spectroscopy, as well as by bromide and phosphorus analysis. Escherichia coli adenylosuccinate synthetase is inactivated by 8-BDB-TGTP at pH 7.0 at 25 degrees C. Pretreatment of the enzyme with N-ethylmaleimide (NEM) blocks the exposed Cys(291) and leads to simple pseudo-first-order kinetics of inactivation. The inactivation exhibits a nonlinear relationship of initial inactivation rate versus 8-BDB-TGTP concentration, indicating the reversible association of 8-BDB-TGTP with the enzyme prior to the formation of a covalent bond. The inactivation kinetics exhibit an apparent K(I) of 115 microM and a k(max) of 0.0262 min(-1). Reaction of the NEM-treated adenylosuccinate synthetase with 8-BDB-[(3)H]TGTP results in 1 mol of reagent incorporated/mol of enzyme subunit. Adenylosuccinate or IMP plus GTP completely protects the enzyme against 8-BDB-TGTP inactivation, whereas IMP or GTP alone provide partial protection against inactivation. AMP is much less effective in protection. The results of ligand protection studies suggest that E. coli adenylosuccinate synthetase may accommodate 8-BDB-TGTP as a GTP analog. The new affinity label may be useful for identifying catalytic amino acid residues of protein proximal to the guanosine ring.

Implication of arginine-131 and arginine-303 in the substrate site of adenylosuccinate synthetase of Escherichia coli by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate.

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial fast-phase inactivation is not affected by the presence of active-site ligands and can be completely eliminated by blocking Cys291 of the enzyme with N-ethylmaleimide (NEM). Reaction of the NEM-treated enzyme with 6-BDB-[32P]TAMP results in 2 mol of reagent incorporated/mol of enzyme subunit. The inactivation kinetics of the slow-phase exhibit an apparent KI of 40.6 microM and kmax of 0.0228 min-1. Active-site ligands, either adenylosuccinate or IMP and GTP, completely prevent inactivation of the enzyme by 6-BDB-TAMP, whereas IMP or IMP and aspartate is much less effective in protection. 6-BDB-TAMP-inactivated enzyme has a 3-fold increase in Km for aspartate with no change in Km for IMP or GTP. Protease digestion of 6-BDB-[32P]TAMP inactivated enzyme reveals that both Arg131 and Arg303 are modified by the affinity-labeling reagent. The crystal structure [Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1996) J. Mol. Biol. 264, 1013-1027] and site-directed mutagenesis [Kang, C., Sun, N., Poland, B. W., Gorrell, A., and Fromm, H. J. (1997) J. Biol. Chem. 272, 11881-11885] of E. coli adenylosuccinate synthetase show that Arg303 interacts with the carboxyl group of aspartate and the 2'-OH of the ribose of IMP and Arg131 is involved in stabilizing aspartate in the active site of the enzyme. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analogue in modifying both Arg131 and Arg303 in the active site of adenylosuccinate synthetase.

Ambiguities in mapping the active site of a conformationally dynamic enzyme by directed mutation. Role of dynamics in structure-function correlations in Escherichia coli adenylosuccinate synthetase.

On the basis of ligated crystal structures, Asn21, Asn38, Thr42, and Arg419 are not involved in the chemical mechanism of adenylosuccinate synthetase from Escherichia coli, yet these residues are well conserved across species. Purified mutants (Asp21 --> Ala, Asn38 --> Ala, Asn38 --> Asp, Asn38 --> Glu, Thr42 --> Ala, and Arg419 --> Leu) were studied by kinetics, circular dichroism spectroscopy, and equilibrium ultracentrifugation. Asp21 and Arg419 are not part of the active site, yet mutations at positions 21 and 419 lower kcat 20- and 10-fold, respectively. Thr42 interacts only through its backbone amide with the guanine nucleotide, yet its mutation to alanine significantly increases Km for all substrates. Asn38 hydrogen-bonds directly to the 5'-phosphoryl group of IMP, yet its mutation to alanine and glutamate has no effect on Km values, but reduces kcat by 100-fold. The mutation Asn38 --> Asp causes 10-57-fold increases in Km for all substrates along with a 30-fold decrease in kcat. At pH 5.6, however, the Asn38 --> Asp mutant is more active, yet binds IMP 100-fold more weakly, than the wild-type enzyme. Proposed mechanisms of ligand-induced conformational change and subunit aggregation can account for the properties of mutant enzymes reported here. The results underscore the difficulty of using directed mutations alone as a means of mapping the active site of an enzyme.

Residues essential for catalysis and stability of the active site of Escherichia coli adenylosuccinate synthetase as revealed by directed mutation and kinetics.

Examined here by directed mutation, circular dichroism spectroscopy, and kinetics are the relationships of five residues, Asp13, Glu14, Lys16, His41, and Arg131, to the catalytic function and structural organization of adenylosuccinate synthetase from Escherichia coli. The D13A mutant has no measurable activity. Mutants E14A and H41N exhibit 1% of the activity of the wild-type enzyme and 2-7-fold increases in the Km of substrates. The mutant K16Q has 34% of the activity of wild-type enzyme and Km values for substrates virtually unchanged from those of the wild-type system. Mutation of Arg131 to leucine caused only a 4-fold increase in the Km for aspartate relative to the wild-type enzyme. The dramatic effects of the D13A, E14A, and H41N mutations on kcat are consistent with the putative roles assigned to Asp13 (catalytic base), His41 (catalytic acid), and Glu14 (structural organization of the active site). The modest effect of the R131L mutation on the binding of aspartate is also in harmony with recent crystallographic investigations, which suggests that Arg131 stabilizes the conformation of the loop that binds the beta-carboxylate of aspartate. The modest effect of the K16Q mutation, however, contrasts with significant changes brought about by the mutation of the corresponding lysines in the P-loop of other GTP- and ATP-binding proteins. Crystallographic structures place Lys16 in a position of direct interaction with the gamma-phosphate of GTP. Furthermore, lysine is present at corresponding positions in all known sequences of adenylosuccinate synthetase. We suggest that along with a modest role in stabilizing the transition state of the phosphotransfer reaction, Lys16 may stabilize the enzyme structurally. In addition, the modest loss of catalytic activity of the K16Q mutant may confer such a selective disadvantage to E. coli that this seemingly innocuous mutation is not tolerated in nature.

A study of Escherichia coli adenylosuccinate synthetase association states and the interface residues of the homodimer.

The state of aggregation of adenylosuccinate synthetase from Escherichia coli is a point of controversy, with crystal structures indicating a dimer and some solution studies indicating a monomer. Crystal structures implicate Arg143 and Asp231 in stabilizing the dimer, with Arg143 interacting directly with bound IMP of the 2-fold related subunit. Residue Arg143 was changed to Lys and Leu, and residue Asp231 was changed to Ala. Matrix-assisted laser desorption ionization mass spectroscopy and analytical ultracentrifugation of the wild-type and the mutant enzymes indicate a mixture of monomers and dimers, with a majority of the enzyme in the monomeric state. In the presence of active site ligands, the wild-type enzyme exists almost exclusively as a dimer, whereas the mutant enzymes show only slightly decreased dissociation constants for the dimerization. Initial rate kinetic studies of the wild-type and mutant enzymes show similar kcat and Km values for aspartate. However, increases in the Km values of GTP and IMP are observed for the mutant. Changes in dissociation constants for IMP are comparable with changes in Km values. Our results suggest that IMP binding induces enzyme dimerization and that two residues in the interface region, Arg143 and Asp231, play significant roles in IMP and GTP binding.

Activity and biospecificity of proteolyzed forms and dimeric combinations of recombinant human and murine nerve growth factor.

Purified recombinant human nerve growth factor (rhNGF) and submaxillary gland-derived murine NGF (muNGF) were characterized by amino acid composition, polyacrylamide gel electrophoresis (PAGE), reversed-phase HPLC (RP-HPLC), and high-performance ion-exchange chromatography (HPIEC). Limited tryptic digest of the N and C termini of the 120-residue form of rhNGF produced a species of 109 residues (10-118). The previously observed natural murine analogue of this variant, muNGF lacking the first eight N-terminal amino acids, was also isolated as a homodimer. Both species were purified using HPIEC and characterized by amino acid analysis, N-terminal sequence, PAGE, and RP-HPLC analysis. Each of the four homodimeric species was evaluated in some or all of the following biological assays for NGF: chick dorsal root and sympathetic ganglion assays and rat pheochromocytoma-12 cell line neurite extension assay. The 118-residue homodimeric versions of both rhNGF and muNGF displayed equivalent bioactivity, whereas the N terminal-modified molecules presented activity reduced by 50- to 100-fold. Utilizing HPIEC, we have examined the ability of the monomeric forms of any two of the homogeneous dimeric species of rhNGF to recombine. We have shown that not only can all of the previously described species form dimers by recombination, but an interspecies dimer can be created between muNGF and rhNGF.

Human recombinant soluble decay accelerating factor inhibits complement activation in vitro and in vivo.

Complement plays a role in activating the inflammatory response and has been implicated in the pathogenesis of some inflammatory diseases. With a view toward controlling unwanted C activation, we evaluated the C regulator, human decay accelerating factor (DAF). Three forms of recombinant DAF were purified from transfected Chinese hamster ovary cells: glycophosphatidylinositol (GPI)-linked membrane DAF (mDAF) extracted from cell membranes; spontaneously shed soluble DAF (sDAF) derived from mDAF; and a novel secreted protein (seDAF), generated by deletion of the signal for GPI attachment. We show that all three molecules inhibit both the classical and alternative pathways of C activation. The following observations indicate that mDAF extracted from Chinese hamster ovary cells reincorporates into RBC membranes via its GPI anchor: 1) cells that are preincubated with mDAF and then washed remain fully protected from C-mediated hemolysis; 2) incubation with phosphatidylinositol-specific phospholipase C abolishes this protection; and 3) sDAF and seDAF, which lack a GPI anchor, do not associate with cell membranes. mDAF is a more potent inhibitor of C-mediated hemolysis than either sDAF or seDAF, suggesting that incorporation into cell membranes greatly enhances the efficiency with which DAF inhibits C activation on the cell surface. In contrast, C activation in the fluid phase is inhibited by sDAF and seDAF, but not by mDAF, possibly due to interference by serum lipoproteins. A reversed passive Arthus reaction in guinea pigs was used to evaluate the ability of recombinant seDAF to inhibit C activation in vivo. When administered at dermal sites, seDAF reduced the severity of immune complex-mediated inflammatory reactions induced by a reversed passive Arthus reaction, as judged by both gross and histologic examination. These data indicate that seDAF may be useful as an anti-inflammatory therapeutic.