Page 27 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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Encyclopedia of Physical Science and Technology EN005G-231 June 15, 2001 20:46

634 Enzyme Mechanisms

∗
from E · Glc · ATP. In this case E · Glc · ATP is approxi- taining an 18 O label in the βγ bridging oxygen pro-
ATP
mately 25% of E T , which requires that k off compete with vides the necessary probe for finding this intermediate
the dissociation of ADP (k ADP ) from E · Glc-6-P · ADP. by means of the process below. In these experiments,
off
In this manner the individual rate constants for hexoki- isotopes are used as labels so that the fate of a particu-
nase were largely determined and the order of substrate lar atom may be followed throughout the course of the
association was verified. reaction.
O O O

Ad O P O P 18 OP O Glu
O O O
O O O O O

E Ad O P O P 18 OP O Glu E Ad O P O P 18 O GluP
O O O O O
O 18 O O
E
E Ad O P O P 18 OP O Glu
O 18 O O
O 18 O O

Ad O P O P 18 OP O Glu
O 18 O O

Isotopic labeling has also been cleverly used to demon- The appearance of 18 O in the nonbridging oxygens of
strate the existence of enzyme-bound intermediates that the β-phosphate can be measured by mass spectrometric
do not readily dissociate into solution. The enzyme glu- and NMR methods. The extent of equilibration is partially
tamine synthetase catalyzes the formation of glutamine inhibited by the presence of ammonia as required if glu-
from ATP and ammonia possibly through a tightly bound tamyl phosphate is a reaction intermediate.
glutamyl phosphate intermediate. Isotopic labeling studies of phosphotranferase reac-
tions culminated in the synthesis of ATP chiral at the
COOH CONH 2
γ -phosphorus. Chirality was achieved by the synthesis of
CH 2 CH 2 16 17 18
ADP [γ - O, O, O]ATP of one configuration, and the anal-
ATP NH 3
CH 2 CH 2 ysis of its chirality was achieved by stereochemically
controlled transfer of the γ -phosphoryl moiety to (S)-
H 2 N COOH H 2 N COOH
propane-1,2-diol where the absolute configuration was
If the formation of glutamyl phosphate were reversible determined by a chemical/mass spectrometric sequence.
and occurred in the absence of ammonia, then the presence The observation of inversion of configuration has been
of a symmetric torsion motion at the cleavage site might accepted as evidence of an “in-line” displacement mech-
be used to anism at phosphorus by the two bound substrates; the ob-
servation of retention of configuration was used to impli-
O O
cate the existence of a phosphoryl enzyme intermediate in

C OH C OPO 3 the phosphoryl transfer process. For hexokinase, our case
study, the finding is one of inversion, consistent with a
CH 2 CH 2
E ATP E ADP direct transfer mechanism.
CH 2 CH 2
D. Triosephosphate Isomerase
H 2 N COOH H 2 N COOH
Triosephosphate isomerase (TIM) catalyzes the inter-
detect an isotopic exchange brought about by glutamyl conversion of D-glyceraldehyde-3-phosphate (G3P) and
18
phosphate formation. The synthesis of γ - O 4 P-ATP con- dihydroxyacetone phosphate (DHAP). The equilibrium