PLENARY LECTURES
luated for some states [9]. It has been found that
this coupling can be ferromagnetic or antiferromagnetic [9].
It is well established now that one iron atom can
be abstracted from many [4Fe-4S] clusters to yield
[3Fe-4S] clusters. Recently it has been suggested
for A. vinelandii ferredoxin I [10] that a sulfur
can be removed to yield a structure with a
[4Fe-3S] core. A similar situation may occur in
C. pasteurianum hydrogenase [11].
(2,
PL5 — WE
BERT L. VALLEE
Center for Biochemical and Biophysical Sciences and Medicine
Harvard Medical School
250 Longwood Avenue, Boston, MA 02115
U.S.A.
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D.O. HEARSHEN, W.R. DUNHAM, E.P. DAY, T.A.
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[2] H. BEINERT, A.J. THOMSON, Arch. Biochem. Biophys.,
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TARR,
KENT,
6
CONCURRENT CRYOKINETIC
AND CRYOSPECTROSCOPIC
CHARACTERIZATION OF INTERMEDIATES
IN METTALLOENZYME ACTION
The detection and definition of intermediates in
reaction pathways, a problem central to the delineation of structure-function relationships in
enzymology, have resisted solution by and large.
We have developed an approach capable of wide
application to such studies through a combination
of cryokinetics with cryospectroscopy. Such cryospectrokinetic studies provide concurrent structural, kinetic, and chemical data on short-lived intermediates in the course of the interactions of
enzymes with their substrates and inhibitors. Subzero temperatures extend the lifetimes of these intermediates and, combined with rapid-mixing and
rapid-scanning instrumentation, allow simultaneous measurement of both their physical-chemical and kinetic characteristics.
Carboxypeptidase A has served as our model
system. The studies have been performed with a
low-temperature stopped-flow instrument which
also serves as a cryospectrometer [1]. The intermediates have been monitored directly through fluorescence generated by radiationless energy transfer
(RET) between enzyme tryptophans and the
dansyl group of enzyme-bound, rapidly hydrolyzed peptide and ester substrates which provide the
basis for measurement of the rates of formation
and breakdown of intermediates. N-Dansylated
oligopeptides and their ester analogues exhibit Michaelis-Menten kinetics over the temperature range —20 to +20°C with kcal/Km values of
(0.3-3) x 10 7 m-1 s ' at + 20°C, pH 7.5. The cryoRev. Port. Quim., 27 (1985)