The reactive cysteine residue within the small subunit of Escherichia coli carbamoyl phosphate synthetase has been identified using the technique of site-directed mutagenesis. Three cysteine residues have previously been found to react with N-ethylmaleimide (NEM) under controlled reaction conditions. Two of these cysteine residues are found on the large subunit, while the third cysteine is located on the small subunit. In the present investigation, Cys-248 of the small subunit has been identified as the residue that reacts with NEM in the presence of MgATP and bicarbonate. Three cysteine residues of the small subunit at positions 131, 214, and 248 were individually mutated to serine residues. These site-specific changes, in addition to N-ethylmaleimide-labeling studies, demonstrated that Cys-248 is the amino acid that reacts with N-ethylmaleimide. Substitution of Cys-248 of the small subunit with larger residues (Asp, Phe, Arg, and Trp) was conducted in order to more closely mimic the observed properties of the NEM-labeled enzyme. The partial glutaminase activity of the C248D mutant increased 40-fold relative to the wild-type enzyme, while the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Similar, but less dramatic, effects were observed for the other mutants, C248S, C248R, C248F, and C248W. There was good correlation between the extent of enhancement of the partial glutaminase activity and an uncoupling of the phosphorylation reactions that occur on the large subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the synthesis of carbamoyl phosphate from bicarbonate, ammonia, and two molecules of MgATP. The enzyme is composed of two nonidentical subunits. The small subunit catalyzes the hydrolysis of glutamine to glutamate and ammonia. The large subunit catalyzes the formation of carbamoyl phosphate and has the binding sites for bicarbonate, ammonia, MgATP, and the allosteric ligands IMP, UMP, and ornithine. The allosteric ligands are believed to bind to the extreme C-terminal portion of the large subunit. Truncation mutants were constructed to investigate the allosteric binding domain. Stop codons were introduced at various locations along the carB gene in order to delete amino acids from the carboxy-terminal end of the large subunit. Removal of 14-119 amino acids from the carboxy-terminal end of the large subunit resulted in significant decreases in all of the enzymatic activities catalyzed by the enzyme. A 40-fold decrease in the glutamine-dependent ATPase activity was observed for the delta 14 truncation. Similar losses in activity were also observed for the delta 50, delta 65, delta 91, and delta 119 mutant proteins. However, formation of carbamoyl phosphate was detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects were observed for UMP with either the delta 91 or delta 119 truncation mutants, but alterations in the catalytic activity were observed in the presence of ornithine even after the removal of the last 119 amino acids from the large subunit of CPS. Six conserved amino acids within the allosteric domain were mutated.(ABSTRACT TRUNCATED AT 250 WORDS)

Retroviral transduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into cells can increase their resistance to the cytotoxic effects of these drugs. We evaluated the ability of wild-type hDHFR and 20 mutant enzymes (13 with single-amino acid substitutions, 7 with two substitutions) to prevent growth inhibition in antifolate-treated CCRF-CEM cells. The wild-type enzyme and all of the variants significantly protected transduced cells from trimetrexate (TMTX)-induced growth inhibition. However, only half of the variants conferred more protection than does the wild-type enzyme. For the variants tested, the observed protective effect was higher for TMTX than for methotrexate (< or =7.5-fold increased resistance), piritrexim (< or =16-fold), and edatrexate (negligible). Transduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (approximately 200-fold). Protection from loss of cell viability was similar to protection from growth inhibition. The protection associated with a particular mutant hDHFR did not result from the level of expression: Efficient protection resulted from low affinity of the variant for antifolates, reasonable catalytic activity, and good thermal stability. Clones isolated from a polyclonal population of transduced cells varied by as much as 30-fold in their resistance to TMTX, the resistance differences depending on hDHFR expression levels.

Three conserved histidine residues, His-243, His-781, and His-788, located within the large subunit of carbamoyl phosphate synthetase from Escherichia coli were identified by sequence identity comparisons. These three histidine residues were individually mutated to asparagine residues. The H243N mutant enzyme was found to be critical for carbamoyl phosphate synthesis as the mutant protein was unable to synthesize carbamoyl phosphate at a significant rate (< 1/1500). By analysis of the effects of this mutation on the partial reactions catalyzed by CPS, it was determined that this mutation blocked the formation of the carbamate intermediate from carboxyphosphate and ammonia. The H781N mutant enzyme had an order of magnitude reduction for both the rate of carbamoyl phosphate formation and ATP synthesis which is consistent with the proposal that the carboxyl-terminal half of the large subunit is primarily involved in the phosphorylation of the putative carbamate intermediate. This mutation also reduced the effects of the allosteric activator ornithine on the Km parameters for ATP in the overall biosynthetic reaction and ADP in the ATP synthesis reaction. The H788N mutant enzyme is a functional protein which maintains the ability to synthesize carbamoyl phosphate at a rate comparable to that of the wild-type enzyme. The effects of this mutation are 10-fold reductions of the ATP synthetase and the bicarbonate-dependent ATPase activities with substantial increases in the Km values for ATP in the full biosynthetic reaction and for ADP in the ATP synthesis reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

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