title: Complexes of aspartate aminotransferase with hydroxylamine derivatives: spectral studies in solution and in the crystalline state.
abstract: Hydroxylamine and its derivatives of general formula H2NOR react with aldehydes and aldimines to produce oximes. If R corresponds to the side chain of a natural amino acid, such compounds can be thought of as analogs of the corresponding amino acids, lacking the alpha-carboxylate group. Oximes formed between such compounds and pyridoxal phosphate in the active site of aspartate amino-transferase mimic external aldimine intermediates that occur during catalysis by this enzyme. The properties of oxime derivatives of mitochondrial aspartate aminotransferase with hydroxylamine and 6 compounds H2NOR were studied by absorption spectroscopy and circular dichroism in solution and by linear dichroism in crystals. Stable oximes, absorbing at lambda max congruent to 380 nm and exhibiting a negative Cotton effect, were obtained with the carboxylate-containing compounds. The oximes formed with carboxylate-free compounds showed somewhat different properties and stability. With H-Tyr a stable complex absorbing at lambda max congruent to 370 nm rather than at 380 nm, was obtained, H-Ala and H-Phe produced unstable oximes with the initial absorption band at lambda max congruent to 380 nm that was gradually replaced by a band at lambda max congruent to 340 nm. The species absorbing at 340 nm were shown to be coenzyme-inhibitor complexes which were gradually released from the enzyme. A similar 330-340 nm absorption band was observed upon reaction of the free coenzyme with all hydroxylamine inhibitors at neutral pH-values. The results of the circular dichroism experiments in solution and the linear dichroism studies in microcrystals of mAspAT indicate that the coenzyme conformation in these inhibitor/enzyme complexes is similar to that occurring in an external aldimine analogue, the 2-MeAsp/mAspAT complex. Co-crystallizations of the enzyme with the H2NOR compounds were also carried out. Triclinic crystals were obtained in all cases, suggesting that the "closed" structure cannot be stabilized by a single carboxylate group.
authors.affiliation: Department of Structural Biology, University of Basel, Switzerland.
authors.forename: L T
authors.initials: LT
authors.lastname: Delbaere
authors.forename: J
authors.initials: J
authors.lastname: Kallen
authors.forename: Z
authors.initials: Z
authors.lastname: Markovic-Housley
authors.forename: A R
authors.initials: AR
authors.lastname: Khomutov
authors.forename: R M
authors.initials: RM
authors.lastname: Khomutov
authors.forename: M Y
authors.initials: MY
authors.lastname: Karpeisky
authors.forename: J N
authors.initials: JN
authors.lastname: Jansonius
mesh_terms: D000818:Animals; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002645:Chickens; D003460:Crystallization; D003461:Crystallography; D006898:Hydroxylamines / Q000138:chemical synthesis / Q000378:metabolism*; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D010091:Oximes / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012996:Solutions; D013057:Spectrum Analysis / Q000379:methods
chemical_list: D006898:Hydroxylamines; D010091:Oximes; D012996:Solutions; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases