Mechanism of 3-methylaspartase probed using deuterium and solvent isotope effects and active-site directed reagents: Identification of an essential cysteine residue

The mechanism of the L-threo-3-methylaspartate ammonia-lyase (EC 4.3.1.2) r eaction has been probed using deuterium and solvent isotope effects with th ree different substrates, (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-3-methylaspartic acid. Each substrate appears to form a covale nt adduct with the enzyme through the amination of a dehydroalanine (DehydA la-173) residue. The true substrates are N-protonated and at low pH, the al kylammonium groups are deprotonated internally in a closed solvent-excluded pocket after K+ ion, an essential cofactor, has become bound to the enzyme . At high pH, the amino groups of the substrates are able to react with the dehydroalanine residue prior to K+ ion binding. This property of the syste m gives rise to complex kinetics at pH 9.0 or greater and causes the format ion of dead-end complexes which lack Mg2+ ion, another essential cofactor. The enzyme-substrate adduct is subsequently deaminated in two elimination p rocesses. Hydrazines act as alternative substrates in the reverse reaction direction in the presence of fumaric acid derivatives, but cause irreversib le inhibition in their absence. Borohydride and cyanide are not inhibitors. N-Ethylmaleimide also irreversibly inactivates the enzyme and labels resid ue Cys-361. The inactivation process is enhanced in the presence of cofacto r Mg2+ ions and Cys-361 appears to serve as a base for the removal of the C -3 proton from the natural substrate, (2S,3S)-3-methylaspartic acid. The de hydroalanine residue appears to be protected in the resting form of the enz yme by generation of an internal thioether cross-link. The binding of the s ubstrate and K+ ion appear to cause a conformational change which requires hydroxide ion. This is linked to reversal of the thioether protection step and generation of the base for substrate deprotonation at C-3. The deaminat ion reaction displays high reverse reaction commitments and independent evi dence from primary deuterium isotope effect data indicates that a thiolate acts as the base for deprotonation at C-3. (C) 1999 Published by Elsevier S cience Ltd. All rights reserved.