th  - 1988 - 51st ENC Conference

INHIBITION OF ALANINE RACEMASE BY THE PHOSHATE ANALOG OF
188 ~LANINE, I-(AMINOETHYL)PHOSPHATE (ALA-P): IDENTIFICATION
~ A SCHIFF BASE LINKAGE IN THE ENZYME-INHIBITOR COMPLEX BY SOLID STATE
-N-NMR. ,
Val~rie Copi~, W. Stephen Faraci, Christopher T. Walsh,
and Robert G. Griffin
Departments of Chemistry and Biology,
and the Francis Bitter National Magnet Laboratory,
Massachusetts Institute of Technology, Cambridge, MA 02139
Alanine racemases are a group of pyridoxal-5'-phosphate (PLP)
containing enzymes which catalyze the racemization of L- and D-alanine,
the latter being an essential component of the peptidoglycan layer of
bacterial cell wall. Although the kinetics of inactivation of alanine
racemases from gram positive bacteria by Ala-P have been well
determined, the structure of the inactive enzyme complex remained to be
determined.
Solid State NMR technique was used to address the issue of whether
or not AIa-P forms a covalent linkage to ~e enzyme's PLP cofactor.
Solid State, Magic Angle Sample Spinning N-NMR experiments combined
with cross-polarization technique were performed at low temperatures on
microcrystals of Ala-P-alanine racemase complex. The NMR results show
that the inactive complex forms a protonated Schiff base to
pyridoxal-5'-phosphate (PLP) in the enzyme's active site. Solid State
NMR spectra of Schiff bases and Ala-Pl~Odel compounds were also
accumulated to provide a database of N-chemical shifts.
Dynamic and Confromational Structure of CORD Factor Glycolipids in
] 8 9 I Model Membranes as Determined by Solid-State 2H NMR:
, r
R. A. Byrd and T. K. Lira, Biophysics Laboratory, Division of Biochemistry and
Biophysics/FDA, 8800 Rockville Pike, Bethesda, MD 20892
As part of our studies of cell surface carbohydrates and their intermolecular interactions,
a recent study has dealt With the structural features of a particular glycolipid. Our studies are
aimed at elucidating the role of these glycolipids in defining the physical and chemical properties
of the mycobacterial cell surface. The glycolipid is a synthetic analog of a natural component
referred to as CORD FACTOR. Certain virulent strains of bacteria form long filaments or
serpentine-like 'cords'. The cord factor has been isolated and characterized as trehalose-6,6'-
dimycolate. Recently, there is renewed interest in its immunostimulant properties and its
antitumor activities, which prompted the testing of several synthetic analogues.
The dynamical behaviors of the trehalose head-group and of the hydrocarbon tails
(dipalmitate and di{2-tetradecylhexadecanoate}) in the liquid-crystalline phase were investigated
by solid-state 2H NMR. Selective synthetic incorporation of 2H on both saccharide rings and the
hydrocarbon chains leads to a complete study of the entire molecule. From the fully assigned 2H
spectra and the respective quadrupole splittings, the conformation of the head group was
determined in a number of systems. Clear evidence exists for pronounced ring strain in the
trehalose moiety. This may have significant implications for understanding the surface
carbohydrate structures such as LPS.
193