Requirement for the Carboxyl-terminal Domain of Saccharomyces cerevisiae Carbamoyl-phosphate Synthetase

Angela L Lim,Susan G Powers-Lee

doi:10.1074/jbc.271.19.11400

Abstract

The arginine-specific carbamoyl phosphate synthetase of Saccharomyces cerevisiae is a heterodimeric enzyme, with a 45-kDa CPA1 subunit binding and cleaving glutamine, and a 124-kDa CPA2 subunit accepting the ammonia moiety cleaved from glutamine, binding all of the remaining substrates and carrying out all of the other catalytic events. CPA2 is composed of two apparently duplicated amino acid sequences involved in binding the two ATP molecules needed for carbamoyl phosphate synthesis and a carboxyl-terminal domain which appears to be less tightly folded than the remainder of the protein. Using deletion mutagenesis, we have established that essentially all of the carboxyl-terminal domain of CPA2 is required for catalytic function and that even small truncations lead to significant changes in the CPA2 conformation. In addition, we have demonstrated that the C-terminal region of CPA2 can be expressed as an autonomously folded unit which is stabilized by specific interactions with the remainder of CPA2. We also made the unexpected finding that, even when ammonia is used as the substrate and there is no catalytic role for CPA1, interaction with CPA1 led to an increase in the Vmax of CPA2 in crude extracts.

Highlights

CPSases function to catalyze the same overall reaction (Equation 1), they differ in various properties as shown in Table I [1, 2]
The specific activities for the indicated transformants were as follows: 0.28 for low copy number pAL6 (CPA1 repressed); 0.37 for pAL6 ϩ chromosomal CPA1 expression; 0.44 for high copy number pRS424/CPA2 (CPA1 repressed); 0.60 for pRS424/CPA2 ϩ chromosomal CPA1 expression; 0.36 for pRS424/CPA2 ϩ pJL113/ST4 grown in arginine to repress CPA1 expression from both the chromosome and the plasmid; and 1.11 for pRS424/CPA2 ϩ pJL113/ST4. It appears that even though ammonia is being used as the substrate in the assay and there is no catalytic role for the amidotransferase activity of 45-kDa CPA1, interaction with this small subunit must affect the conformation of the 124-kDa CPA2 and thereby increase the rate of carbamoyl phosphate synthesis
Our analysis of C-terminal deletions in yeast CPA2 has demonstrated that essentially all of domain D is required for full catalytic function

Summary

EXPERIMENTAL PROCEDURES

Materials—Oligonucleotides were synthesized at the Tufts University Analytical Core Facility. General Procedures—All molecular biological procedures, including agarose gel electrophoresis, restriction enzyme digestion, ligation, and bacterial transformation, were performed essentially as described by Sambrook et al [30]. Crude extracts for electrophoretic and Western blot analysis were prepared as described above for enzymatic assay except that the following lysis buffer was used in order to minimize proteolysis: 50 mM potassium phosphate, 2 mM EDTA, 10% glycerol, 1 mM ⑀-aminocaproic acid, 0.77 ␮M aprotinin, 2 mM benzamidine, 50 ␮M p-chloromercuriphenylsulfonate, 1.65 ␮M chymostatin, 6.3 ␮M leupeptin, 5 mM ␤-mercaptoethanol, 4.2 mM Pefabloc, 21.9 ␮M pepstatin, and 1 mg/ml phenanthroline, final pH 7.4. Did not alter the original BamHI site; the resulting 1318-base pair PCR fragment was digested with NcoI and BamHI and ligated into the corresponding sites of the pET11-d polylinker region; site-directed mutagenesis, using unique site elimination [23], was used to reverse the base change (A to G) at the ϩ4 position of CPA2 that had been necessary to introduce the NcoI site, yielding the plasmid pET11-d/NB; the BamHI fragment containing the remaining sequence of wild-type CPA2 (1256 –3357 base pairs) was cloned from pAL6 into the BamHI site of pET11-d/NB to yield pET11-d/CPA2

RESULTS

DISCUSSION

Methods