Structure at 2.9-A resolution of aspartate carbamoyltransferase complexed with the bisubstrate analogue N-(phosphonacetyl)-L-aspartate.

Abstract

In an x-ray diffraction study by the isomorphous replacement method, the structure of the complex of aspartate carbamoyltransferase (EC 2.1.3.2) bound to the bisubstrate analogue N-(phosphonacetyl)-L-aspartate has been solved to 2.9-A resolution (R = 0.24). The large quaternary structural changes previously deduced by molecular replacement methods have been confirmed: the two catalytic trimers (c3) move apart by 12 A and mutually reorient by 10°, and the regulatory dimers (r2) reorient each about its twofold axis by about 15°. In this, the T-to-R transition, new 3). The structure consists of catalytic (c) and regulatory (r) chains in a (c3)2(r2)3 oligomer which has D3 symmetry ( Fig. 1) (4-6). Previous x-ray diffraction studies have established the structure of the less active T form to a resolution of 2.6 A (7,8). Also, the large changes in quaternary structure that occur in the transition to the R form as the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA) is bound have been described (9). Less precise indications of these large conformational changes have been established by other investigators (10-13). Our refinement of this R form proceeded so slowly that we have undertaken an independent solution of the R structure bound to PALA by the method of multiple isomorphous replacement (MIR). The results, described below, establish the binding site of PALA and elucidate previously undescribed changes in tertiary structure of the catalytic chains, and they also enable us to look C6 FIG. 1. Subunit and domain structure of aspartate carbamoyltransferase, as viewed down the threefold axis. Domains of the catalytic (c) chains C1-C6 are equatorial (eq) and polar (po), while those of the regulatory (r) chains R1-R6 are zinc (zn) and allosteric (al).at the T-to-R homotropic transition as typified by these two x-ray structures: unligated-and PALA-aspartate carbamoyltransferase. MATERIALS AND METHODSCrystal Growth. The enzyme was isolated and purified (14) from regulatory mutant E. coli provided by J. C. Gerhart and grown at the New England Enzyme Center. Crystals were obtained by dialyzing for 1-2 weeks at 21°C against 50 mM maleic acid/1 mM PALA/3 mM NaN3/N-ethylmorpholine to pH 5.9 and conductivity 3.4 inS. Temperature control was critical for crystallization. After growth, the crystals were transferred, still within their microdialysis vials, to 20% (wt/vol) polyethylene glycol-6000/crystallization buffer at pH 5.8. 1643The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The subunit interfaces of oligomeric enzymes are conserved to a similar extent to the overall protein sequences

“…The percentage of identity for these positions versus the overall identity percentage for each pair of sequences from N. K Grishin and M.A . Phillips (1982), Ke et al (1984), Krause et al (1985), Kim et al (1987), Gouaux and Lipscomb (1988), Kantrowitz and Lipscomb (1988), Ke and Lipscomb (1988). References for TIM: Banner et al (1975), Wierenga et al (1987, , .…”

Shared active sites in oligomeric enzymes: model studies with defective mutants of aspartate transcarbamoylase produced by site-directed mutagenesis.

“…Isolated C trimers are readily dissociated into folded, inactive monomers upon treatment with NaSCN, and subsequent reconstitution studies have shown that the regeneration of enzyme activity is coincident with formation of the C trimer (6). Crystallographic studies of liganded and unliganded ATCase (7,8) and hybridization experiments with C subunits (5) have led to the Abbreviations: ATCase, aspartate transcarbamoylase; C trimer, catalytic trimer; Cxxx, C trimer with identity of mutated residue in the polypeptide chain designated by X subscript; subscript L, Lys …”

Stucture of anticancer antibiotic L-alanosine

“…The crystal structure of L-aspartate carbamoyltransferase, an enzyme of the pyridine nucleotide synthesis, known to utilize L-alanosine as an analogous substrate (Gale et al, 1968;Anandaraj et al, 1980) has been published (Honzatko, Crawford, Monaco, Ladner, Ewards, Evans, Warren, Wiley, Ladner & Lipscomb, 1982), but the site for L-aspartate binding could not be determined unequivocally from these studies. More recently, the crystal structure of the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate with this enzyme was reported (Krause, Volz & Lipscomb, 1985). The dihedral angle for the C(2)-C(3) bond in this structure is 110 °.…”