Aspartate Transcarbamoylase from Escherichia coli : Electron Density at 5.5 Å Resolution

Abstract

The allosteric enzyme, aspartate transcarbamoylase (EC 2.1.3.2), has previously been shown in our x-ray diffraction studies to have D3-32 symmetry. There are six catalytic (C) and six regulatory (R) chains in the molecular complex (R6C6). Our three-dimensional x-ray diffraction study of this enzyme (R32, a = 131 A, c = 200 A) at 5.5 A resolution shows a spatial arrangement of the two catalytic trimers C3 above and below an equa- Regulation of enzyme activity is an important biochemical process within living cells. A number of mechanisms have been considered: (a) steric and conformational effects associated with the direct binding of small inhibitors at or near the active site, (b) the direct covering of the active site by another protein, (c) change of conformation at the active site upon binding of an effector in another region of the enzyme molecule, and (d) a conformational change transmitted from one protein molecule to another in an enzyme complex. We present here a 5.5 A resolution structure of aspartate transcarbamoylase, features of which suggest a new possible component to general mechanisms of regulation: namely, the direct steric regulation of substrate access to the active site in a central cavity of the molecular complex.Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis (1, 2), the reaction of carbamoylphosphate with aspartate to yield carbamoylaspartate and phosphate. The activity of ATCase is modulated by allosteric effectors. Cytidine triphosphate, an end-product of the pyrimidine pathway, is a feedback inhibitor (3, 4), while ATP stimulates ATCase. These effectors thus provide one mechanism for achieving balance between pyrimidines and purines for biosynthesis of nucleic acids.The catalytic and regulatory functions of ATCase (molecular weight 310,000, ref. 5) are associated with different polypeptide chains C and R, respectively (4, 5). The hexameric nature of ATCase (R6C6) was established by (a) the presence of elements of both 2-fold and 3-fold molecular symmetry in the x-ray study (6) and (b) the molecular weights of C and R, along with sequence analysis of the R chain (7). Dissociation of ATCase by reaction of the R-chain thiols with mercurials (5) has been interpreted (6, 7) to yield catalytic trimers C3 and regulatory dimers R2. The relative positions of the C3 units above and below a wider equatorial belt of three regulatory dimers were established in the x-ray study (8), which also indicated the presence of a large solvent-filled region in the center of the ATCase molecule. We describe here a more detailed view of this -and other features of the electron density of ATCase at 5.5-A resolution from a-greatly improved map. METHODSThe crystal form has space group R32, and hexagonal axes a 131 A and c = 200 A. The molecule is situated at the intersection of a 3-fold and three 2-fold symmetry axes ifl the crystal. Hence, one-sixth of the molecule is in the crystallographic asymmetric unit.Following our initial attempts to solve for the m...

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Genetics and biochemistry of carbamoyl phosphate biosynthesis and its utilization in the pyrimidine biosynthetic pathway.

“…The three-dimensional structure of the enzyme appears to consist of two catalytic trimers separated by three regulatory dimers (27,155). In the center of the molecule is a large cavity which appears to be accessible for active sites (44,153). Access to this cavity seems to be via six channels near the regulatory chains, a finding which has led to the speculation that the regulatory mechanism may involve modulation of access of substrates through these channels (153).…”

A kinetic model of cooperativity in aspartate transcarbamylase

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