Active site aspartate transcarbamylase

Type A zinc sites have been identified in metallothioneins (31) and aspartate transcarbamylase (32). Both involve four sulfur atoms at 2.33 A from the zinc. Such sites will not be catalytically active, as the coordination sphere of the metal is saturated. Therefore, a function for the zinc as a pivot, holding together the regulatory chains of the enzyme in a firm but flexible manner, is proposed (32). 

Figure 7. Interactions at the active site of aspartate transcarbamylase (ATCase). N-phosphonoacetyl-L-asparate (PALA) is a bisubstrate analog of the two natural substrates of ATCase, carbamyl phosphate and L-aspartate. PALA is shown bound in the active site of ATCase. Noncovalent interactions between PALA and side-chains of the protein are shown as dashed lines. Specific residues are indicated by their one letter abbreviation and by their position in the protein sequence (e.g., HI 34 = histidine at position 134). The active site is composed of residues from two separate polypeptide chains (denoted by primed and unprimed residue numbers). Note the complimentarity of the site and the ligand. The same interactions are used to align and catalyze the condensation of ATCase s natural substrates (Monaco et al., 1978).

Site-directed mutagenesis has been used to establish that the active site lies at the interface between subunits of certain oligomeric enzymes (62-64). The analysis relies on restoration of activity on forming a hybrid from proteins containing mutations at two positions. Studies of this type were first performed on aspartate transcarbamylase (aspartate carbamoyltransferase) (62, 63), where an active hybrid catalytic trimer was isolated from a mixture of two inactive mutants. The rationale for this analysis is shown in Fig. 8, illustrating wodc done on ribulose-bisphosphate carboxylase (64). Two mutant enzymes, eaeh unable to carry out catalysis, were recombined to form hybrids. Based on random association of monomers to form the catalytic dimer as shown in Fig. 8, it is expected that 50% of the trimers should form one wild-type active site (B, C), such that the mixture of the hybrids exhibits 25% of the wild-type activity. This complementation demonstrates that the active site must be at the interface between the subunits. 

Fig. is. Model for the mechanism of homotropic cooperativity in aspartate transcarbamylase. Shown schematically are the two extreme conformations in the T state and the R state. The binding of the substrates at one active site induces the domain closure in that catalytic chain and requires a quaternary conformational change which allows the 240s loops of the upper and lower catalytic chains to move to their final positions. The formation of the R state, in a concerted fashion, is further stabilized by a variety of new interactions as shown. [Reprinted with permission from Ref. (/).]... 

Data taken form Craik et al. (1987) for rat trypsin Wilks et al. (1990) for B. stereothermophilus lactate dehydrogenases Muraki et al. (1992) for human lysozyme in which the active site residues refer to E35, D53, W64, D102 and W109 and AGr = -RTIn[(koat/Kra)niutant/ (kcat/Kra)wiidl Stebbins and Kantrowitz (1992) for B. subtilis aspartate transcarbamylase Casal et al. (1987) for yeast triosephosphate isomerase Dupureur et al. (1992) for pancreatic phospholipase Mrabet et al. (1992) for Actinoplanes missouriensis xylose isomerase. 

Sometimes the action of an enzyme is controlled by a substance other than the substrate or products. These enzymes are called allosteric enzymes. The site on the enzyme molecule that reacts with the allosteric control molecule is different from the active reaction site of the enzyme. The separate allosteric control sites and active sites have been observed by x-ray diffraction in the enzyme aspartate transcarbamylase, an enzyme of molecular mass 310,000 d which consists of six subunits. 

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