The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 5 Dioxygenation

Figure 5.1 Intramolecular dioxygenases (both atoms of O 2 into same molecule) Different types of aromatic cleavages for catechol dioxygenases contain nonheme Fe III (EPR) red contain nonheme Fe II (no EPR) colorless Intermolecular dioxygenases (two O atoms of O 2 go into two different products) Dioxygenases Incorporation of both atoms of O 2 into substrates

Scheme 5.1 Criegee rearrangement [1,2] shift to peroxide Mechanism is based on model studies and crystal structures A Mechanism for Intradiol Dioxygenases Using Protocatechuate 3,4-Dioxygenase as an Example

Scheme 5.2 Alternate Mechanism Criegee dioxetane Product found to have lost some 18 O (not from washout) Criegee versus dioxetane mechanisms for intradiol dioxygenases Catechol 1,2-dioxygenase This would have retained both 18 O atoms **

Scheme 5.3 nucleophilic addition  to C-O Criegee Evidence for Criegee as in Scheme 5.2 A Mechanism for Extradiol Dioxygenases Using 2,3- Dihydroxyphenylpropionate 1,2-Dioxygenase as an Example

Scheme 5.4 Both diastereomers give the same ratio of 5.23 and 5.24 Consistent with a reversible cyclopropylcarbinyl radical opening Evidence for a Radical Intermediate in Extradiol Catechol Dioxygenases “Reversible”

Scheme 5.5 Heme-dependent enzyme arachidonic acid (with double bond) cyclooxygenase activity peroxidase activity Reactions Catalyzed by Prostaglandin H Synthase (Cyclooxygenase)

Scheme 5.6 Conversion of Prostaglandin H to Other Prostaglandins

Scheme 5.7 Mixture of 18 O O 2 gives both ring O’s as 18 O or 16 O; therefore from same molecule of O 2 H’s at C-8, C-11, C-12 retained H at C-9 lost in PGE, and retained in PGF 1  Only pro-S H at C-13 removed 3 18 O atoms Prostaglandin H Synthase-catalyzed Incorporation of 18 O 2 into 8,11,14-Eicostrienoic Acid

Scheme 5.8 Therefore, endoperoxidation occurs prior to hydroperoxidation at C-15 isolated as a byproduct A Hydroxylated By-product Isolated from the Prostaglandin H Synthase-catalyzed Reaction

No PGE formed from this Further Evidence that Endoperoxidation Occurs First

Scheme 5.9 Tyr-385 Evidence for pathway a: intermediate found containing one O 2 and no 3 H Tyr385Phe has peroxidase activity, but no cyclooxygenase activity Two Possible Mechanisms for Prostaglandin H Tyr-385 Peroxidase active site is adjacent to cyclooxygenase active site - peroxidase regenerates protein radical in cyclooxygenase ( X) and it reduces PGG to PGH - contains ferric heme Iron-oxo species from heme initially abstracts H from Tyr-385 ( X-H)

Scheme 5.10 no C-9 H C-9 H retained Conversion of Prostaglandin H to Prostaglandin E and F  

Scheme 5.11 Intermolecular Dioxygenases nonheme Fe 2+ like heme enzymes if  -ketoglutarate, then this is succinic acid keeps iron reduced usually  -ketoglutarate Reaction catalyzed by  -keto acid-dependent dioxygenases

Scheme 5.12 Mechanism to Account for Formation of Succinic Acid, CO 2, and an Iron-oxo Species from CD spectrum like heme-oxo species Conversion of Fe II and O 2 to a high energy iron-oxo species catalyzed by  -keto acid-dependent dioxygenases

Scheme 5.13 Early Mechanisms for Hydroxylation of Prolyl Residues by Prolyl Hydroxylase (proposed in 1979)  - ketoglutarate

Scheme 5.14 clavulanic acid Mechanism for Clavaminate Synthase- Catalyzed Ring Closure

Scheme 5.15 Evidence for iron-oxo mechanism D isotope effect w/CD 3 Reactions resemble heme-dependent enzymes Reactions Catalyzed by Thymine Hydroxylase

gets epoxidizedS-oxygenationhydroxylation N-dealkylation All similar to heme-dependent P450 reactions With 18 O O in succinate 1 18 O in product Other Alternative Substrates for Thymine Hydroxylase

Scheme 5.16 inactivates thymine hydroxylase, like P450 carbene insertion from NMR after tryptic digestion Inactivation of Thymine Hydroxylase by 5-Ethynyluracil

Scheme 5.17 Intramolecular Version of  -Keto Acid Dioxygenase O atom partial exchange with solvent 1 18 O atom Reaction catalyzed by (p-hydroxyphenyl)pyruvate dioxygenase

Scheme 5.18 Model Study for the Mechanism of (p-Hydroxyphenyl)pyruvate Dioxygenase

If 5.56 is incubated with the enzyme, no product is formed Enzyme also catalyzes sulfoxidations like P450 R = H or F are substrates Evidence Against this Mechanism

Scheme 5.19 exchange with solvent Proposed Mechanism for (p-Hydroxyphenyl)pyruvate Dioxygenase Mechanism more like heme-dependent enzymes Like heme peroxide