The Organic Chemistry of Enzyme-Catalyzed Reactions
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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 l.jpg
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 l.jpg
[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 l.jpg

[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 l.jpg
An “Overshoot” Experiment with (R)-(-)-glutamate to Test for a Two-base Mechanism for Glutamate Racemase

in D2O

Figure 9.1


Slide6 l.jpg

Another Test for a Two-Base Mechanism

Elimination of HCl from threo-3-chloroglutamic acid by the C73A and C184A mutants for glutamate racemase

Scheme 9.2


Proposed mechanism for proline racemase l.jpg
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 l.jpg
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 l.jpg
Pyridoxal 5-Phosphate (PLP) Dependent Racemases

Proposed mechanism for PLP-dependent alanine racemase

Scheme 9.4

Usually, a one-base mechanism


How can plp enzymes catalyze selective bond cleavage l.jpg

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?


Slide11 l.jpg
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 l.jpg
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 l.jpg
Other Racemases to

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 l.jpg
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 l.jpg

H297N Mutant Capable of Elimination of HBr from ( for the Deuterium Solvent Exchange Results. 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 l.jpg

Epimerases for the Deuterium Solvent Exchange Results.

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 l.jpg
-Cleavage Mechanism for Peptide Epimerization for the Deuterium Solvent Exchange Results.

Mechanism 2

Scheme 9.9

10 mM NH2OH has no effect on product formation

Therefore, mechanism 2 is unlikely.


Deprotonation reprotonation mechanism l.jpg
Deprotonation/Reprotonation Mechanism for the Deuterium Solvent Exchange Results.

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 l.jpg

Epimerization with Redox Catalysis for the Deuterium Solvent Exchange Results.

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 l.jpg

UDP-Glucose 4-Epimerase for the Deuterium Solvent Exchange Results.

UDP-glucose

UDP-galactose

In H218O, no incorporation of 18O into product

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


Slide21 l.jpg
Tritium is incorporated from NAD for the Deuterium Solvent Exchange Results. 3H 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 l.jpg
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 l.jpg
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 l.jpg
Mechanistic Pathway for the GDP- Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto IntermediateD-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 l.jpg

[1,2]-H Shift Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

Reaction catalyzed by aldose-ketose isomerases

Lobry de Brun-Alberda von Ekenstein Reaction

Scheme 9.16


Cis enediol mechanism for aldose ketose isomerases l.jpg

Two Mechanisms Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

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 l.jpg
Hydride transfer mechanism for aldose-ketose isomerases Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

Mechanism 2

Scheme 9.18

Partial incorporation of solvent observed - inconsistent with hydride mechanism


Reaction catalyzed by phenylpyruvate tautomerase l.jpg

[1,3]-H Shift Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

Enolization

Reaction catalyzed by phenylpyruvate tautomerase

removes pro-R hydrogen

Scheme 9.19


Conformations of phenylpyruvate that would form z and e enols by phenylpyruvate tautomerase l.jpg

Two Conformers Possible Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

Conformations of phenylpyruvate that would form Z- and E-enols by phenylpyruvate tautomerase

Scheme 9.20


To test for favored conformation l.jpg
To Test for Favored Conformation Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

favored inhibitors

Therefore syn geometry to E enol most likely


Carbanion mechanism for allylic isomerases l.jpg

Allylic Isomerizations Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

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 l.jpg
Carbocation mechanism for allylic isomerases Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

Mechanism 2

Scheme 9.22

This H comes from solvent, not from the substrate


1 3 sigmatropic hydride shift mechanism for allylic isomerases l.jpg
[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 l.jpg

Carbanion Mechanism isomerases

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 l.jpg
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 l.jpg

Kinetic Competence of Enol by 3-Oxo-

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

Scheme 9.27

same rates



Slide38 l.jpg
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 l.jpg

Carbanion Mechanism the Active Site of 3-oxo-

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

Scheme 9.29


One mechanism for the function of asp 99 in the active site of 3 oxo 5 steroid isomerase l.jpg

Asp-99 Located Adjacent to Tyr-14 the Active Site of 3-oxo-

One mechanism for the function of Asp-99 in the active site of 3-oxo-5-steroid isomerase

Scheme 9.30


Slide41 l.jpg
Crystal structure with equilenin bound is consistent with Asp-99 and Tyr-14 both coordinated to oxyanion

equilenin


4 oxalocrotonate tautomerase l.jpg
4-Oxalocrotonate Tautomerase Asp-99 and Tyr-14 both coordinated to oxyanion

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 l.jpg

Carbocation Mechanism Asp-99 and Tyr-14 both coordinated to oxyanion

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 l.jpg
Evidence for a Carbocation Mechanism Asp-99 and Tyr-14 both coordinated to oxyanion

Ki = 14 pM

rate is 1.8  10-6 times

transition state analogue inhibitor


Proposed mechanism for isopentenyl diphosphate isomerase l.jpg
Proposed Mechanism for Isopentenyl Diphosphate Isomerase Asp-99 and Tyr-14 both coordinated to oxyanion

Scheme 9.35


Aza allylic isomerization l.jpg
Aza-allylic Isomerization Asp-99 and Tyr-14 both coordinated to oxyanion

Scheme 9.36


Reaction catalyzed by l aspartate aminotransferase l.jpg

PLP-dependent Aminotransferase Asp-99 and Tyr-14 both coordinated to oxyanion

Reaction catalyzed by L-aspartate aminotransferase

Scheme 9.37


First half reaction catalyzed by aspartate aminotransferase l.jpg
First Half Reaction Catalyzed by Aspartate Aminotransferase Asp-99 and Tyr-14 both coordinated to oxyanion

Scheme 9.38

PMP


Second half reaction catalyzed by aspartate aminotransferase l.jpg
Second Half Reaction Catalyzed by Aspartate Aminotransferase Asp-99 and Tyr-14 both coordinated to oxyanion

Scheme 9.39

This is the reverse of the mechanism in Scheme 9.38


Crystal structures of l.jpg
Crystal structures of: Asp-99 and Tyr-14 both coordinated to oxyanion

  • 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 l.jpg
Evidence for Quinonoid Intermediate Asp-99 and Tyr-14 both coordinated to oxyanion

pseudosubstrate

quinonoid form observed at 490 nm


Slide52 l.jpg
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 l.jpg

Cis-Trans Catalyzed by Many PLP-dependent Aminotransferases 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 l.jpg
Proposed Mechanism for the Reaction Catalyzed by Maleylacetoacetate Isomerase

Scheme 9.42


Reaction catalyzed by phosphoglucomutases l.jpg

Phosphate Isomerization Maleylacetoacetate Isomerase

Reaction catalyzed by phosphoglucomutases

only -anomer binds

Scheme 9.45


Proposed mechanism for the reaction catalyzed by phosphoglucomutases l.jpg

Native State of Enzyme is Phosphorylated Maleylacetoacetate Isomerase

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



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