Abstract
S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography. The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence. These are the first residues to be implicated in the activity and/or structure of the enzyme. N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer. Accumulation of the dimer parallels the loss of the enzyme activity. When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-[ethyl-2-3H] maleimide was present predominantly in the dimer. Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M. Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M. Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.
Highlights
N-Ethylmaleimide-modified S-adenosylmethionine synthetase existsmainly as a dimerin conditions where the native enzyme is a tetramer
Data for protein standards show that the B, 0.22 mM enzyme subunits were incubated with 0.9 mM N-[ethylstandard errors for most amino acids are
Effects of ligands onrate of N-ethylmakimide modification of S-adenosylmethionine synthetase the dissociation of AdoMet synthetase into subunits at protein con- Reactions were initiated by addition of NEM andwere monitored centrations below 0.8 p~ subunits, 50 mM Tris/Cl was substituted by the decrease in the absorbance of NEM a t 305 nm
Summary
Are the first residues to be implicated in the activity andlor structure of the enzyme. Upon modification the strengthof dimer-dimer interactions disiminished into the protein was monitored by incubation of enzyme with [‘HI NEM for periods between 10 s and 15 min in 50 mM Hepes/KOH, 50 mM KC1,lO mM MgClz. Aliquots of the reaction mixture were diluted 25-fold into 10mM dithiothreitol in mM Hepes/KOH, 50 mM KCl. From the diluted samples, aliquots were removedfor assay of enzyme activity. This article must be hereby marked “aduer- The solution of modified AdoMet synthetase was adjusted to 8 M tisement” in accordance with 18U.S.C. Section 1734solelyto indicate urea and 10 mM iodoacetic acid by addition of solid reagents. Data for protein standards show that the B, 0.22 mM enzyme subunits were incubated with 0.9 mM N-[ethylstandard errors for most amino acids are
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