Reactions of plant copper/topaquinone amine oxidases with N6–aminoalkyl derivatives of adenine

Plant copper/topaquinone-containing amine oxidases (CAOs, EC 1.4.3.6) are enzymes oxidising various amines. Here we report a study on the reactions of CAOs from grass pea (Lathyrus sativus), lentil (Lens esculenta) and Euphorbia characias, a Mediterranean shrub, with N6-aminoalkyl adenines representing combined analogues of cytokinins and polyamines. The following compounds were synthesised: N6-(3-aminopropyl)adenine, N6-(4-aminobutyl)adenine, N6-(4-amino-trans-but-2- enyl)adenine, N6-(4-amino-cis-but-2-enyl)adenine and N6-(4-aminobut-2-ynyl)adenine. From these, N 6-(4-aminobutyl) adenine and N6-(4-amino-trans-but-2-enyl)adenine were found to be substrates for all three enzymes ðKm , 1024MÞ: Absorption spectroscopy demonstrated such an interaction with the cofactor topaquinone, which is typical for common diamine substrates. However, only the former compound provided a regular reaction stoichiometry. Anaerobic absorption spectra of N6-(3-aminopropyl)adenine, N6-(4-amino-cis-but-2-enyl)adenine and N6-(4-aminobut-2-ynyl)adenine reactions revealed a similar kind of initial interaction, although the compounds finally inhibited the enzymes. Kinetic measurements allowed the determination of both inhibition type and strength; N 6-(3-aminopropyl)adenine and N6-(4-amino-cis-but-2- enyl)adenine produced reversible inhibition ðKi , 1025 –1024MÞ whereas, N 6-(4-aminobut-2-ynyl)adenine could be considered a powerful inactivator

A Simple Method to Determine Electrospray Response Factors of Noncovalent Complexes

The quantitative study of noncovalent complexes by electrospray mass spectrometry requires the determination of the relative response of each species. The method proposed here to determine the electrospray response factors is based on the use of (1) an internal standard and (2) the mass balance equation applied to one binding partner M, for which different complexes MxLy are detected in the electrospray mass spectra. A set of experiments providing various ratios between the complexes (e.g. different ligand concentrations in a titration experiment or different time points in a kinetics experiment) is used to generate a set of independent linear equations that can be solved using simple matrix algebra to find the response factors of each MxLy complex relative to that of the internal standard. The response factors can then be used to determine equilibrium dissociation constants or for the quantitative monitoring of reaction kinetics. The first is illustrated with a study of DNA-ligand complexes, where we show that neither minor groove binding nor intercalation dramatically affects the DNA response factor. The second is illustrated with a study of the association kinetics of the telomeric G-quadruplex dGGG(TTAGGG)3 with its complementary strand, where the response factors allow correcting for the relative response of the quadruplex and the long duplex and obtaining reproducible association rate constants independently of the source tuning potentials

Intriguing mass spectrometric behavior of guanosine under low energy collision-Induced dissociation: H2O adduct formation and gas-phase reactions in the collision cell

An in-depth study of the fragmentation pathway of guanosine was conducted by using an in-source collision-induced dissociation high-mass accuracy tandem mass spectrometry experiment. The equivalent of MS4 data, a level of information normally achieved on ion trap instruments, was obtained on a Q-TOF mass spectrometer. The combination of the features of high-resolution, accuracy, and in-source CID permitted the unambiguous elucidation of the different fragmentation pathways. Furthermore the elemental compositions of the product ions generated were assigned and their mutual genealogical relationships established. Formerly proposed dissociation pathways of guanosine were revisited and elaborated on more deeply. Furthermore, the presence of H2O in the collision cell of several tandem MS instruments was demonstrated and its effect on the product ion spectra investigated. The neutral gain of H2O by particular fragments of guanosine was experimentally proven by using argon, saturated with H218O, as the collision gas. Data indicating the occurrence of more complex reactions in the collision cell as a result of the presence of H2O were produced, specifically relating to neutral gain/neutral loss sequences. In silico calculations supported the experimental observation of neutral gain by guanosine fragments and predicted a similar behavior for adenosine. The latter was subsequently experimentally confirmed

Calcium-Induced Structural Transitions of the Calmodulin−Melittin System Studied by Electrospray Mass Spectrometry: Conformational Subpopulations and Metal-Unsaturated Intermediates

Calmodulin (CaM) is a calcium-sensing protein that can bind to and activate various target enzymes. Here, electrospray ionization mass spectrometry (ESI-MS) was used to investigate calcium-induced structural changes of CaM, as well as binding to the model target melittin (Mel). Nonspecific metalation artifacts were eliminated by conducting the experiments in negative ion mode and with calcium tartrate as metal source [Pan et al. (2009) Anal. Chem. 81, 5008]. Two coexisting CaM subpopulations can be distinguished on the basis of their ESI charge state distributions, namely, relatively disordered conformers (CaM(D), high charge states) and more tightly folded proteins (CaM(F), low charge states). Calcium titration experiments on isolated CaM reveal that the transition from apo-CaM(D) to Ca(4).CaM(F) proceeds with apparent K(d) values of 10, 14, 30, and 12 microM. In the presence of Mel, a gradual [Ca(2+)] increase results in an overall population shift from apo-CaM(D) to Ca(4).CaM(F).Mel. This transition involves various intermediates, Ca(n).CaM(F).Mel with n = 0, ..., 3, as well as apo-CaM(D).Mel. Thus, neither the binding of four Ca(2+) nor the existence of a tightly folded CaM conformation is a prerequisite for target binding. Millisecond time-resolved ESI-MS experiments were conducted to monitor the response of a premixed CaM-Mel solution to a calcium concentration jump, thereby mimicking the conditions encountered in a cellular signaling context. The resulting data suggest that the formation of Ca(4).CaM(F).Mel proceeds along three parallel kinetic pathways: (1) metal binding to CaM(D) followed by formation of a compact protein-target complex, (2) folding of the apoprotein, then target binding, followed by metal complexation, (3) target binding to apo-CaM(D) followed by sequential metal binding. The exact structural properties of the various metal-unsaturated CaM species, as well as their physiological roles, remain to be elucidated