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The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 9 Isomerizations. Isomerizations. Conversion of one molecule into another with the same formula Hydrogen shifts to the same carbon: [1,1]-H shift Hydrogen shifts to the adjacent carbon: [1,2]-H shift
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The Organic Chemistry of Enzyme-Catalyzed ReactionsChapter 9Isomerizations
Not PLP - no visible absorbance
Not pyruvoyl - acid hydrolysis gave no pyruvate
No M2+ - EDTA has no effect
No acyl intermediates - no 18O wash out of [C18O2H]Glu
Not oxidation/reduction - 2H is incorporated into C-2 from 2H2O
Therefore deprotonation/reprotonation mechanism
Amino acid racemases(A) One-base mechanism for racemization (epimerization), (B) Two-base mechanism for racemization (epimerization)
One base: substrate proton transferred to product
Two base: incorporated proton from solvent
With Glu racemase: solvent deuterium in product, not substrate
also, primary kinetic isotope effect with [2-2H]Glu
Inactivation by ICH2COO- only after a reducing agent is added
(RSH or NaBH4)
Reduces active site disulfide to dithiol
Because substrates bind tightest at the transition state of the reaction, a compound resembling the TS‡ structure would be more tightly bound
TS‡ analogue inhibitor for Pro racemase
Proposed mechanism for PLP-dependent alanine racemase
Usually, a one-base mechanism
PLP was a coenzyme for decarboxylases (break C-COOH bond) and now for racemases (break C-H bond)How can PLP enzymes catalyze selective bond cleavage?
all sp2 + p atoms
The -bond that is parallel to (overlapping with) the p-orbitals will break (C-H in this case)
The rectangles represent the plane of the pyridine ring of the PLP.
The angle of viewing is that shown by the eye in Figure 9.2.
pyridine ring of PLP
The -charge stops free rotation, which results in selective bond cleavage
Reaction catalyzed by mandelate racemase
No internal return in either direction
With (R)-mandelate no -H exchange with solvent
With (S)-mandelate there is exchange with solvent
Lys-166 acts on the (S)-isomer, and His-297 acts on the (R)-isomer
no solvent exchange
H297N mutant is capable of exchanging the -H of the S-isomer, but not the R-isomer
H297N Mutant Capable of Elimination of HBr from (S)-9.5, but not from the (R)-isomerElimination of HBr from (S)-p-(bromomethyl)mandelate, catalyzed by the H297N mutant of mandelate racemase
K166R mutant catalyzes elimination of HBr from the (R)-isomer, but not from the (S)-isomer
Peptide epimerasesElimination/addition (dehydration-hydration) mechanism for peptide epimerization
With 18O in the Ser OH group, no loss of 18O as H218O
Therefore, mechanism 1 is unlikely.
10 mM NH2OH has no effect on product formation
Therefore, mechanism 2 is unlikely.
In D2O D is incorporated into product, not substrate (short incubation; monitored by electrospray ionization mass spectrometry)
Deuterium isotope effect for [-2H]-peptides in the L- to D-direction
is different from that in the D- to L-direction
These results are consistent with mechanism 3.
two different enzymes
C-H cleavage at C-3 and C-5 show kinetic isotope effects (3.4 and 2.0, respectively)
In 2H2O 2H incorporation at both C-3 and C-5
Partial exchange gives only C-3 proton exchange, never only C-5 proton exchange (ordered sequential mechanism)
In H218O, no incorporation of 18O into product
No change in oxidation state, but is deprotonation/reprotonation reasonable?
The enzyme requires NAD+; no exchange with solvent
Evidence for 9.14:
incubate enzyme with UDP-galactose,quench with NaB3H4. 3H at C-4 of both UDP-glucose and UDP-galactose
No change in oxidation state, but NAD+ required
suprafacial transfer of H
Partial incorporation of solvent observed - inconsistent with hydride mechanism
Therefore syn geometry to E enol most likely
This H comes from solvent, not from the substrate
Unlikely -- [1,3]-hydride shift is allowed antarafacial,
which is geometrically impossible
from NOE studies
To probe the function of Tyr-14
Uv spectrum bound to enzyme is same as neutral amine.
Therefore Tyr-14 does
not protonate C-3 carbonyl
Structure bound to enzyme even at low pH (pKa of the phenol must be very low).
H bonds to dienolate
From deuterated substrates, substrate analogues, and reactions run in D2O, 9.42 to 9.44 is suprafacial
No exchange of solvent into substrate, only into product
One base mechanism
Ki = 14 pM
rate is 1.8 10-6 times
transition state analogue inhibitor
This is the reverse of the mechanism in Scheme 9.38
All are consistent with mechanisms in Schemes 8.39 and 9.38
quinonoid form observed at 490 nm
-H is transferred to the CH2 of PMP suprafacially; therefore one-base mechanism
-2H removed from si-face and delivered to pro-S CH2 of PMP
GSH acts as a coenzyme, not as a reducing agent
No 2H incorporated into substrate or product from 2H2O