Asparagine synthetase B catalyzes the assembly of asparagine from aspartate, Mg2+ATP, and glutamine. Here, we describe the three-dimensional structure of the enzyme from Escherichia coli determined and refined to 2.0 Å resolution. Protein employed for this study was that of a site-directed mutant protein, Cys1Ala. Large crystals were grown in the presence of both glutamine and AMP. Each subunit of the dimeric protein folds into two distinct domains. The N-terminal region contains two layers of antiparallel β-sheet with each layer containing six strands. Wedged between these layers of sheet is the active site responsible for the hydrolysis of glutamine. Key side chains employed for positioning the glutamine substrate within the binding pocket include Arg 49, Asn 74, Glu 76, and Asp 98. The C-terminal domain, responsible for the binding of both Mg2+ATP and aspartate, is dominated by a five-stranded parallel β-sheet flanked on either side by α-helices. The AMP moiety is anchored to the protein via hydrogen bonds with Oγ of Ser 346 and the backbone carbonyl and amide groups of Val 272, Leu 232, and Gly 347. As observed for other amidotransferases, the two active sites are connected by a tunnel lined primarily with backbone atoms and hydrophobic and nonpolar amino acid residues. Strikingly, the three-dimensional architecture of the N-terminal domain of asparagine synthetase B is similar to that observed for glutamine phosphoribosylpyrophosphate amidotransferase while the molecular motif of the C-domain is reminiscent to that observed for GMP synthetase.
P37, an outer-membrane bacterial protein from Mycoplasma hyorhinis, is a molecule whose presence on the surface of many tumor cells correlates highly with increased neoplastic invasivity and metastasis. P37 was overexpressed in Escherichia coli, purified by affinity chromatography and crystallized. Useful single crystals for X-ray diffraction structural studies have been grown by oil-immersion methods from a solution of 40% PEG 4000, 0.1 M ammonium bromide in a 0.1 M citrate buffer at pH 4.0. X-ray diffraction data were collected at the F2 beamline at CHESS with a crystal-to-CCD detector distance of 150 mm, collecting 1° oscillation slices with an exposure time of 30 s per frame. A 212° sweep of data (99.8% completeness) were collected from a single crystal under cryoconditions, with a maximal useful diffraction pattern to 1.8 Å resolution. The crystals are shown to be monoclinic and have been assigned to space group P21, with unit-cell parameters a = 50.02, b = 67.26, c = 59.89 Å, β = 108.29° and a scaling Rsym of 0.076 for 34 882 unique reflections. Packing considerations indicate that there is one molecule per asymmetric unit. It is expected that in the near future the structure of p37 will be obtained using phases from traditional heavy-atom isomorphous replacement and/or halide-soak methods. Elucidation of the structure of p37 may be paramount to producing new antibody-based anticancer therapeutic agents.
Abstract Evidence is presented in support of the hypothesis that the pyruvate dehydrogenase multienzyme complex of intact rabbit heart mitochondria may be regulated by a phosphorylation-dephosphorylation mechanism. Mitochondria incubated in the presence of pyruvate plus l-malate and either ADP or uncoupler exhibit nearly identical, rapid rates of pyruvate oxidation but possess markedly different ATP levels. It was shown that under metabolic conditions which lead to a release or mobilization of intramitochondrial magnesium and which also have a high intramitochondrial ATP level, pyruvate oxidation was nearly completely inhibited after a brief lag phase. It was shown that the addition of exogenous magnesium to uncoupled mitochondria supplemented with ADP caused a more rapid inhibition of pyruvate oxidation. The observed inhibition of pyruvate oxidation was dependent upon the time of preliminary incubation with uncoupler and ADP and was atractyloside-sensitive. Evidence was obtained indicating that the inhibition of pyruvate oxidation was specific for the substrate, pyruvate, i.e. the oxidation of l(-)palmitylcarnitine or α-ketoglutarate was unaffected under conditions leading to an inhibition of pyruvate oxidation. In addition, it was demonstrated that the inhibition of pyruvate oxidation was not caused by an accumulation of either NADH or acetyl-CoA, both inhibitory products of the pyruvate dehydrogenase reaction, during the course of these experiments. The experiments reported in this communication indicate that the control of the availability of both ATP and magnesium are crucial for the regulation of the pyruvate dehydrogenase multienzyme complex. These studies are consistent with the possibility that the pyruvate dehydrogenase-linked protein kinase may be an effective means of regulating the conversion of pyruvate to acetyl-CoA in intact metabolic systems such as the isolated mitochondrion.
Asparagine synthetase catalyzes the ATP-dependent formation of l-asparagine from l-aspartate and l-glutamine, via a β-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that l-cysteine sulfinic acid competitively inhibits this enzyme by interfering with l-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of β-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799−5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B.
In experiments aimed at determining the mechanism of nitrogen transfer in purF amidotransferase enzymes, 13C and 15N kinetic isotope effects have been measured for both of the glutamine-dependent activities of Escherichia coli asparagine synthetase B (AS-B). For the glutaminase reaction catalyzed by AS-B at pH 8.0, substitution of heavy atom labels in the side chain amide of the substrate yields observed values of 1.0245 and 1.0095 for the amide carbon and amide nitrogen isotope effects, respectively. In the glutamine-dependent synthesis of asparagine at pH 8.0, the amide carbon and amide nitrogen isotope effects have values of 1.0231 and 1.0222, respectively. We interpret these results to mean that nitrogen transfer does not proceed by the formation of free ammonia in the active site of the enzyme and probably involves a series of intermediates in which glutamine becomes covalently attached to aspartate. While a number of mechanisms are consistent with the observed isotope effects, a likely reaction pathway involves reaction of an oxyanion with β-aspartyl-AMP. This yields an intermediate in which C−N bond cleavage gives an acylthioenzyme and a second tetrahedral intermediate. Loss of AMP from the latter gives asparagine. An alternate reaction mechanism in which asparagine is generated from an imide intermediate also appears consistent with the observed kinetic isotope effects.
