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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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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
  • Hydrogen shifts to two carbon atoms away: [1,3]-H shift
1 1 hydrogen shift racemase with no cofactors glutamate racemase
[1,1]-Hydrogen ShiftRacemase with no cofactorsGlutamate racemase

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

slide4

[1,1]-Hydrogen Shift

Amino acid racemases

(A) One-base mechanism for racemization (epimerization), (B) Two-base mechanism for racemization (epimerization)

Scheme 9.1

One base: substrate proton transferred to product

Two base: incorporated proton from solvent

With Glu racemase: solvent deuterium in product, not substrate

(B)

also, primary kinetic isotope effect with [2-2H]Glu

an overshoot experiment with r glutamate to test for a two base mechanism for glutamate racemase
An “Overshoot” Experiment with (R)-(-)-glutamate to Test for a Two-base Mechanism for Glutamate Racemase

in D2O

Figure 9.1

proposed mechanism for proline racemase
Proposed Mechanism for Proline Racemase

Scheme 9.3

Inactivation by ICH2COO- only after a reducing agent is added

(RSH or NaBH4)

Reduces active site disulfide to dithiol

transition state analogue inhibitor
Transition State Analogue Inhibitor

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

resembles 9.3

pyridoxal 5 phosphate plp dependent racemases
Pyridoxal 5-Phosphate (PLP) Dependent Racemases

Proposed mechanism for PLP-dependent alanine racemase

Scheme 9.4

Usually, a one-base mechanism

slide11
Stereochemical Relationship Between the -Bonds Attached to C and the p-Orbitals of the -System in a PLP-Amino Acid Schiff Base

Figure 9.2

PLP

all sp2 + p atoms

The -bond that is parallel to (overlapping with) the p-orbitals will break (C-H in this case)

dunathan hypothesis for plp activation of the bonds attached to c in a plp amino acid schiff base
Dunathan Hypothesis for PLP Activation of the Bonds Attached to C in a PLP-Amino Acid Schiff Base

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

Figure 9.3

The -charge stops free rotation, which results in selective bond cleavage

other racemases
Other Racemases

Reaction catalyzed by mandelate racemase

Scheme 9.5

No internal return in either direction

With (R)-mandelate no -H exchange with solvent

With (S)-mandelate there is exchange with solvent

slide14
A Two-base Mechanism for Mandelate Racemase that Accounts for the Deuterium Solvent Exchange Results.

Lys-166 acts on the (S)-isomer, and His-297 acts on the (R)-isomer

solvent exchange

no solvent exchange

H297N mutant is capable of exchanging the -H of the S-isomer, but not the R-isomer

Scheme 9.6

slide15

H297N Mutant Capable of Elimination of HBr from (S)-9.5, but not from the (R)-isomer

Elimination of HBr from (S)-p-(bromomethyl)mandelate, catalyzed by the H297N mutant of mandelate racemase

Scheme 9.7

K166R mutant catalyzes elimination of HBr from the (R)-isomer, but not from the (S)-isomer

elimination addition dehydration hydration mechanism for peptide epimerization

Epimerases

Peptide epimerases

Elimination/addition (dehydration-hydration) mechanism for peptide epimerization

Mechanism 1

Scheme 9.8

With 18O in the Ser OH group, no loss of 18O as H218O

Therefore, mechanism 1 is unlikely.

cleavage mechanism for peptide epimerization
-Cleavage Mechanism for Peptide Epimerization

Mechanism 2

Scheme 9.9

10 mM NH2OH has no effect on product formation

Therefore, mechanism 2 is unlikely.

deprotonation reprotonation mechanism
Deprotonation/Reprotonation Mechanism

Mechanism 3

Scheme 9.10

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

(two-base mechanism)

These results are consistent with mechanism 3.

slide19

Epimerization with Redox Catalysis

Proposed mechanism for dTDP-L-rhamnose synthase-catalyzed conversion of dTDP-4-keto-6-deoxy-D-glucose (9.9) to dTDP-L-rhamnose (9.10)

Scheme 9.11

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)

slide20

UDP-Glucose 4-Epimerase

UDP-glucose

UDP-galactose

In H218O, no incorporation of 18O into product

No change in oxidation state, but is deprotonation/reprotonation reasonable?

slide21
Tritium is incorporated from NAD3H into a derivative of the suspected intermediate of the UDP-glucose 4-epimerase-catalyzed reaction

The enzyme requires NAD+; no exchange with solvent

without OH

reverse reaction

proposed intermediate

Scheme 9.12

proposed mechanism for reaction catalyzed by udp glucose 4 epimerase
Proposed Mechanism for Reaction Catalyzed by UDP-Glucose 4-Epimerase

Scheme 9.13

Evidence for 9.14:

incubate enzyme with UDP-galactose,quench with NaB3H4. 3H at C-4 of both UDP-glucose and UDP-galactose

slide23
Mechanism to Account for Transfer of Hydrogen from the Top Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

Scheme 9.14

slide24
Mechanistic Pathway for the GDP-D-mannose-3,5-epimerase-catalyzed Conversion of GDP-D-mannose (9.15) to GDP-L-galactose (9.18)

No change in oxidation state, but NAD+ required

Scheme 9.15

reaction catalyzed by aldose ketose isomerases

[1,2]-H Shift

Reaction catalyzed by aldose-ketose isomerases

Lobry de Brun-Alberda von Ekenstein Reaction

Scheme 9.16

cis enediol mechanism for aldose ketose isomerases

Two Mechanisms

cis-Enediol mechanism for aldose-ketose isomerases

Mechanism 1

cis-enediol

suprafacial transfer of H

Scheme 9.17

hydride transfer mechanism for aldose ketose isomerases
Hydride transfer mechanism for aldose-ketose isomerases

Mechanism 2

Scheme 9.18

Partial incorporation of solvent observed - inconsistent with hydride mechanism

reaction catalyzed by phenylpyruvate tautomerase

[1,3]-H Shift

Enolization

Reaction catalyzed by phenylpyruvate tautomerase

removes pro-R hydrogen

Scheme 9.19

to test for favored conformation
To Test for Favored Conformation

favored inhibitors

Therefore syn geometry to E enol most likely

carbanion mechanism for allylic isomerases

Allylic Isomerizations

Carbanion mechanism for allylic isomerases

Mechanism 1

This H could come from the substrate (if no solvent exchange)

Scheme 9.21

carbocation mechanism for allylic isomerases
Carbocation mechanism for allylic isomerases

Mechanism 2

Scheme 9.22

This H comes from solvent, not from the substrate

1 3 sigmatropic hydride shift mechanism for allylic isomerases
[1,3]-Sigmatropic hydride shift mechanism for allylic isomerases

Mechanism 3

Scheme 9.23

Unlikely -- [1,3]-hydride shift is allowed antarafacial,

which is geometrically impossible

reaction catalyzed by 3 oxo 5 steroid isomerase

Carbanion Mechanism

Reaction catalyzed by 3-oxo-5-steroid isomerase

Scheme 9.24

Principal reaction transfers 4-H to 6-position; therefore suprafacial

Eliminates carbocation mechanism and [1,3] hydride shift

evidence for an enol intermediate in the reaction catalyzed by 3 oxo 5 steroid isomerase
Evidence for an Enol Intermediate in the Reaction Catalyzed by 3-Oxo-5-steroid Isomerase

Scheme 9.26

further evidence for an enol intermediate in the reaction catalyzed by 3 oxo 5 steroid isomerase

Kinetic Competence of Enol

Further evidence for an enol intermediate in the reaction catalyzed by 3-oxo-5-steroid isomerase

Scheme 9.27

same rates

slide38
Reactions Designed to Investigate the Function of Tyr-14 at the Active Site of 3-oxo-5-steroid Isomerase

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).

Scheme 9.28

Therefore Tyr-14

H bonds to dienolate

slide39

Carbanion Mechanism

Mechanism for suprafacial transfer of the 4-proton to the 6-proton of steroids catalyzed by 3-oxo-5-steroid isomerase

Scheme 9.29

slide41
Crystal structure with equilenin bound is consistent with Asp-99 and Tyr-14 both coordinated to oxyanion

equilenin

4 oxalocrotonate tautomerase
4-Oxalocrotonate Tautomerase

Scheme 9.32

From deuterated substrates, substrate analogues, and reactions run in D2O, 9.42 to 9.44 is suprafacial

(one-base mechanism)

reaction catalyzed by isopentenyl diphosphate isomerase

Carbocation Mechanism

Reaction catalyzed by isopentenyl diphosphate isomerase

isopentenyl diphosphate

dimethylallyl diphosphate

Scheme 9.33

No exchange of solvent into substrate, only into product

One base mechanism

evidence for a carbocation mechanism
Evidence for a Carbocation Mechanism

Ki = 14 pM

rate is 1.8  10-6 times

transition state analogue inhibitor

second half reaction catalyzed by aspartate aminotransferase
Second Half Reaction Catalyzed by Aspartate Aminotransferase

Scheme 9.39

This is the reverse of the mechanism in Scheme 9.38

crystal structures of
Crystal structures of:
  • native enzyme with PLP bound
  • substrate reduced onto PLP
  • enzyme with PMP bound

All are consistent with mechanisms in Schemes 8.39 and 9.38

evidence for quinonoid intermediate
Evidence for Quinonoid Intermediate

pseudosubstrate

quinonoid form observed at 490 nm

slide52
Stereochemistry of Proton Transfer in the First Step Catalyzed by Many PLP-dependent Aminotransferases

-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

pro-S

Scheme 9.40

reaction catalyzed by maleylacetoacetate isomerase

Cis-Trans Isomerization

Reaction catalyzed by maleylacetoacetate isomerase

Scheme 9.41

GSH acts as a coenzyme, not as a reducing agent

No 2H incorporated into substrate or product from 2H2O

proposed mechanism for the reaction catalyzed by maleylacetoacetate isomerase
Proposed Mechanism for the Reaction Catalyzed by Maleylacetoacetate Isomerase

Scheme 9.42

proposed mechanism for the reaction catalyzed by phosphoglucomutases

Native State of Enzyme is Phosphorylated

Proposed mechanism for the reaction catalyzed by phosphoglucomutases

tightly bound

Scheme 9.46

Overall retention of configuration at phosphate

Double inversion

Shown as associative, but could be dissociative