Mechanisms of enzyme
and coenzyme action; stereochemistry and mechanisms of phospho- and
nucleotidyl transferase action; structure and function of multienzyme
complexes
The central thrust of my
research is the elucidation of the mechanisms of enzymatic reactions.
Two aspects of the field of mechanistic enzymology are particularly
interesting to me, and are the basis for ninety percent of my research:
a)
The question of how enzymes utilize binding interactions directed to
nonreacting parts of substrate molecules to catalyze the chemical
transformations of the reacting parts of substrates is one principal
focus of my research. These interactions provide the energy for the
structural transition of enzymes into active conformations. Statements
that are commonly advanced to explain enzymatic catalysis by the active
conformation of an enzyme include those in which the enzyme is
postulated to stabilize transition states or to destabilize ground
states in enzyme-substrate complexes, or both. These are very general
statements that do not explicitly account for the actions of particular
enzymes. A specific description of catalysis, in both structural and
dynamic terms, is needed for a few enzymes. Serine proteases,
isomerases, and the enzymes of galactose metabolism are subjects of my
research in this field; and,
b)
The second focus of my research is the elucidation of the mechanisms of
enzymatic reactions that are so obscure in chemical terms that no
obvious chemical precedent is available. These enzymes depend upon
cofactors about which little is known. Lysine 2,3-aminomutase is one
example of such an enzyme, and it is one subject of my current research
in this field. Lysine 2,3-aminomutase catalyzes the 1,2-amino group
migration in the interconversion of L-lysine and L-beta-lysine. The
required cofactors include pyridoxal-5'-phosphate (PLP),
S-adenosylmethionine, and an iron-sulfur cluster. PLP normally
functions to stabilize carbanions in enzymatic reactions; however, in
the lysine 2,3-aminomutase reaction it appears to facilitate the
rearrangement of a substrate radical. S-Adenosylmethionine is normally
a biological alkylating agent; however, in the lysine 2,3-aminomutase
reaction it functions as a hydrogen transfer agent. Analogous vitamin B12-dependent
aminomutases are also under investigation. The chemical interactions of
these cofactors and the mechanisms by which they function in catalysis
are under investigation. The detailed structure of the enzyme and the
chemistry by which it interacts with cofactors are also under intensive
investigation by use of physical, chemical and biological methods.