Mapping the mechanism-based modification sites in L-aspartase from Escherichia coli.

Inactivation of the enzyme L-aspartase from Escherichia coli by the substrate analog aspartate beta-semialdehyde has previously been shown to occur by the mechanism-based conversion to the corresponding product aldehyde, followed by covalent modification of cysteine-273 (F. Giorgianni et al. (1995) Biochemistry 34, 3529). Inactivation by the product analog, fumaric acid aldehyde (FAA), has now been examined directly by adding a reduction step to the modification protocol in order to stabilize the resulting enzyme-FAA derivative(s). HPLC and mass spectrometric analyses of proteolytic digests of inactivated L-aspartase have confirmed the modification at cysteine-273, and have also identified an additional modified peptide. The inactivation at this additional site involves a crosslink between cysteine-140 and an adjacent lysine. Site-directed mutagenesis studies have shown that cysteine-140 is a very reactive and accessible nucleophile that is not, however, directly involved in enzyme activity. The adjacent lysine-139 that is modified does appear to play a role in substrate binding. A double mutant in which both of the reactive cysteines have been replaced is almost completely insensitive to modification by these substrate and product analogs.

Characterization of neutral fragments in tandem mass spectrometry: a unique route to mechanistic and structural information.

The neutral species eliminated upon fragmentation of fast-moving mass-selected ions can be directly identified by collisional ionization and detection in neutral fragment reionization (Nf R) mass spectra. Establishment of the identity of neutral fragments yields valuable insight into the decomposition mechanism of a precursor ion, as demonstrated for fullerene and alkali metal iodide cluster ions as well as metal ion adducts of amino acids. In addition, neutral fragment reionization also provides structural information that may not be available from the complementary ionic fragments alone; this is illustrated in the differentiation of isomeric mononucleotides. The parameters influencing the appearance of Nf R spectra are discussed and the scope and general applicability of the method are briefly evaluated.

Elimination of the sensitivity of L-aspartase to active-site-directed inactivation without alteration of catalytic activity.

The catalytic activity of the enzyme L-aspartase from Escherichia coli has previously been shown to be sensitive to sulfhydryl reagents. The use of group-specific reagents, and a sequence homology comparison study among the fumarase-aspartase family of enzymes, has not, however, lead to the identification of a specific, essential cysteinyl residue. We have recently shown that L-aspartate-beta-semialdehyde is an alternative substrate for L-aspartase, producing fumaric acid semialdehyde (FAA) which specifically inactivates the enzyme [Schindler, J. F., & Viola, R. E. (1994) Biochemistry 33, 9365]. Proteolytic digests of the resulting inactivated enzyme have now been mapped by HPLC and mass spectrometry. A specific residue (Cys-273) has been determined to be the site of FAA modification. Site-directed mutagenesis of this cysteine in the E. coli enzyme has produced altered enzymes which are considerably less sensitive to active-site-directed inactivation, while retaining full catalytic activity. Thus, cysteine-273 has been identified as an active-site nucleophile that, while not directly involved in catalysis in L-aspartase, is poised to attack an activated double bond in an enzyme-bound product analogue.

Internal energy distributions of tungsten hexacarbonyl ions after neutralization-Reionization.

The internal energy distributions P(ε) transferred to W(CO) 6 (+·) during the kiloelectronvolt collisions that occur upon neutralization-reionization (NR) have been estimated based on the relative abundances of the W(CO) 0-6 (+·) products present in NR spectra (thermochemical method). The average internal energy of the incipient {W(CO) 6 (+·) }(*) ions arising after near thermoneutral neutralization with trimethylamine followed by reionization with O2 is ∼9 eV for 8-keV precursor ions and is mainly deposited during reionization, For comparison, the mean internal energy of {W(CO) 6 (+·) }(*) after electron ionization (EO or collisionally activated dissociation (CAD) is ∼ 6 eV. Making the neutralization step endothermic slightly increases the overall excitation gained; however, a large increase in endothermicity (> 16 eV) causes only a modest rise of the average internal energy (<2 eV). The P(ε) curve for NR increases exponentially up to ∼ 6 eV and levels off at higher energies.. showing that the probability of imparting large internal energies (6-17 eV) is high. In sharp contrast, the most probable excitation on CAD is ≤6 eV, and the probability of deposition of larger energies declines exponentially. The mean internal energies after CAD and NR decrease steadily when the kinetic energy is lowered. The structure (minima-maxima) observed in the P(ε) distribution for El, which most likely originates from Franck-Condon factors, is not reproduced in the distributions for NR or high energy CAD, despite the fact that all three methods involve electronic excitation. Because of the large internal energies transferred upon NR, NR mass spectrometry could be particularly useful in the differentiation of ionic isomers with high dissociation but low isomerization thresholds.