The reactivity of thiol groups and the subunit structure of aldolase

Abstract

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-(14)C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5'-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.

Amino acid sequence of the N‐terminal 158 residues of rabbit muscle aldolase

“…Earlier only two tryptic peptides containing carboxymethyl cysteine could be isolated and identified in fragment CB1, peptides T16 and T13a [6,8,9]. Later we isolated from the tryptic digest of fragment CB1 an additional, carboxymethyl cysteine-containing peptide, T9a, which did not contain lysine or arginine [7].…”

“…As for the N-terminal cyanogen bromide fragment (CB1) only the sequence of some smaller segments were known, that of the N-terminal tryptic peptide [4,5] and the sequence or composition of the cysteine-containing peptides [6][7][8][9].…”

Amino acid sequence homology in the active site of rabbit and sturgeon muscle aldolases

“…During the course of this work and also of a study of the reactivity of the thiol groups of rabbit muscle aldolase [3], it became apparent that there were slight discrepancies between our results and the amino acid sequence around the substrate-binding lysine residue of the rabbit muscle enzyme previously established elsewhere [4,5]. We have therefore reinvestigated that sequence and present it here in comparison with the sturgeon enzyme.…”

“…In fact, the sequence reported here would explain why trypsin fails to cleave at Lys-15 in peptide RX3, since it is well known that Lys-Pro bonds are not normally susceptible to tryptic attack. Moreover, it would also explain the presence of the peptide derived from positions 18-28 in tryptic digests of the intact S-carboxymethylated enzyme, an anomaly to which we have already drawn attention [3], on the reasonable supposition that the Asn-Met bond at position 17-18 is susceptible to chymotryptic-like cleavage during tryptic digestion.…”

Amino acid sequence of a fragment of rabbit muscle aldolase

“…Four out of seven cysteinyl residues of the protein are included in this region of the molecule. One of the four cysteinyl residues, Cys-231, has been found to be most reactive towards bromoacetate but its alkylation did not impair enzymic activity [11][12][13]. In the sequential vicinity of this Cys residue, the substrate-binding Lys-221 is located.…”