The Organic Chemistry of Enzyme-Catalyzed ReactionsChapter 7Carboxylations
CarboxylationsGeneral Concepts • A carbanion (or carbanionic character) must be generated where carboxylation is to occur. Must be a stabilized carbanion. • Metal ion complexation of the oxygen atom of the keto and • enol forms can increase the acidity of an adjacent C-H bond • by 4-6 orders of magnitude • CO2 is an excellent electrophile for carboxylation, • but at physiological pH, it is in low concentration • Predominant form is bicarbonate (HCO3-), which is actually • a nucleophile • To convert bicarbonate into an electrophile, it must be • activated either by phosphorylation or dehydration
In general, all enzymes utilize CO2 except for phosphoenolpyruvate carboxylase and the biotin-dependent enzymes, which use bicarbonate • To determine which is the substrate: • Put CO2 into the enzyme reaction at a concentration • approximating its Km value, and incubate with sufficient • enzyme so that a significant amount of product is produced • in the first few seconds. There are two possible outcomes • (Figure 7.1, next slide):
Carboxylations Test for whether CO2 or HCO3- is the substrate for a carboxylate CO2 + H2O H2CO3 (equilibrium ~ 1 min) electrophile nucleophile Possible outcomes when CO2 is added to a carboxylase Figure 7.1 Also, repeat in the presence of carbonic anhydrase (catalyzes hydrolysis of CO2 H2CO3)
CO2 as Carboxylating Agent Reaction catalyzed by PEP carboxykinase Scheme 7.1 oxaloacetate PEP If run in H218O with CO2, no 18O in products (need large amount of enzyme so no nonenzymatic conversion of CO2 to HCO3-) Addition of [14C]pyruvate does not give [14C]oxaloacetate. Pyruvate or enolpyruvate are not free intermediates.
PEP carboxykinase-catalyzed reaction of PEP with ADP (no CO2) In the absence of CO2, the enzyme acts like a kinase (H+ in place of CO2) pyruvate Scheme 7.2
Reduction of Oxaloacetate by Malate Dehydrogenase If the carboxylase reaction is run in D2O in the presence of malate DH/NADH, no D is in the malate; therefore no enol of oxaloacetate formed. oxaloacetate malate Scheme 7.3 Malate dehydrogenase traps oxaloacetate to prevent nonenzymatic enolization.
Hypothetical Mechanism for PEP Carboxykinase that Involves the Enolate of Oxaloacetate This mechanism is excluded by the previous result: Scheme 7.4
Stereochemistry of the Reaction Catalyzed by PEP Carboxykinase Running the reaction in reverse Scheme 7.5 inversion of stereochemistry Excludes covalent catalytic mechanism
Inconsistent with a double-inversion mechanism for PEP carboxykinase This Mechanism is Excluded: Scheme 7.6
Possible Mechanism for PEP Carboxykinase Concerted mechanism for PEP carboxykinase (or stepwise without release of intermediates) Scheme 7.7
Reaction Catalyzed by Phosphoenolpyruvate Carboxytransphosphorylase (oxaloacetate) Scheme 7.8 Same as PEP carboxykinase except Pi instead of nucleotide diphosphate All mechanistic experiments are the same for the two enzymes
Alkene stereochemistry nomenclature rules for (Z)-1-bromo-1-propene (7.8) Stereochemical Rules Needed to Determine Stereochemistry of PEP Carboxytransphosphorylase re re Figure 7.2
Alkene Nomenclature Rules for (E)-1-bromo-1-propene (7.9) si Figure 7.3 re si-re or re-si? Cite the side with the highest priority group (in this case, Br) Front face is named re-si face
Two Possible Stereochemical Outcomes for Carboxylation of PEP Catalyzed by PEP Carboxytransphosphorylase (Z)-[3-3H]PEP anti-elimination With (E)-[3-3H]PEP, 98% 3H in fumarate; therefore carboxylation from si-re face anti-elimination fumarate observed 98% loss as 3H2O P-O bond of PEP breaks, but C-O bond of PEP breaks with EPSP synthase Scheme 7.10
Vitamin K Cycle for Carboxylation of Proteins blood-clotting proteins binds Ca2+ Scheme 7.11
Calcium-dependent Binding of Clotting Proteins to Cell Surfaces Figure 7.4 -proteases Holds the proteases to the appropriate cells, triggering the blood-clotting cascade
Test for Carbanion vs. Radical Mechanisms for Vitamin K Carboxylase Scheme 7.12 erythro- and threo- erythro- F- elimination, but not threo-; therefore stereospecific (carbanion)
Stereochemical Outcome of Vitamin K Carboxylase-catalyzed Carboxylation of (2S,4R-fluoroglutamate) carboxylation with inversion of stereochemistry Scheme 7.13
Proposed Vitamin K Carboxylase-catalyzed Carboxylation of Glutamate Residues via a Carbanionic Intermediate Scheme 7.14 But where does vitamin K fit into the mechanism?
Model Study for Function of Vitamin K Chemical model study for the activation of vitamin K1 as a base Not a strong enough base to deprotonate 7.20 Model for reduced vitamin K Reaction does not work in absence of O2 strong base Dieckmann condensation Scheme 7.15 Base Strength Amplification Mechanism
Two Proposed Mechanisms for Activation of Vitamin K1 as a Base (not 1O2) Scheme 7.16 When run in 18O2, 0.95 mol atom 18O in epoxide 0.17 mol atom 18O in quinone oxygen
To Determine Which Ketone is Involved Incubation in 16O2 atmosphere gives loss of 0.17 mol atom 18O from 7.23, none from 7.24 Therefore, the ketone next to the methyl group is involved in the reaction
Modified Base Strength Amplification Mechanism for Vitamin K Carboxylase To account for much loss of 18O from substrate Scheme 7.18
Bicarbonate as the Carboxylating Agent Reaction catalyzed by PEP carboxylase PEP No H218O formed (high enzyme concentration, short time at alkaline pH) Scheme 7.19 Therefore HCO3-, not CO2
Concerted (A), Stepwise Associative (B), and Stepwise Dissociative (C) Mechanisms for PEP Carboxylase Note: nucleophilic mechanisms Scheme 7.20 concerted stepwise associative stepwise dissociative No partial exchange detected ([14C]pyruvate does not give [14C]PEP) Therefore, either concerted or intermediate not released
Evidence for Stepwise Mechanism inversion in H218O concerted is suprafacial sigmatropic; therefore retention Also, rate is independent of pH, but the carbon isotope effect for H13CO3- decreases with increasing pH. Not possible with concerted Evidence for dissociative mechanism: Using methyl PEP and HC18O3- more than 1 18O in Pi and substrate recovered has 18O in nonbridging position of phosphate; therefore reversible CO2 + Pi formed (see next slide)
Mechanism for Incorporation of 18O into Substrate C Non-bridging 18O Scheme not in text (after Scheme 7.20) Note: the ultimate carboxylating agent is CO2
Biotin-dependent Enzymes Multisubunit enzymes Covalent attachment of d-biotin to an active site lysine residue Scheme 7.24 Enzyme reactions with HC18O3- give Pi with one 18O and product with 2 18O atoms (bicarbonate)
Reactions Catalyzed by Biotin-dependent Carboxylases Figure 7.5 Diagnostic method for biotin - add avidin KD = 1.3 10-15 M
Mechanism of Biotin-Dependent Carboxylases Partial exchange reaction of 32Pi into ATP (in absence of substrate) with biotin-dependent carboxylases Scheme 7.25 No substrate or product needed Suggests ATP activates bicarbonate
Mechanism for Partial Exchange of 32Pi into ATP with Biotin-dependent Carboxylases Scheme 7.26
Partial Exchange Reaction of [14C]ADP into ATP with Biotin-dependent Carboxylases Scheme 7.27
substrate, HCO3- ATP, M2+ [14C]substrate [14C]product Mechanism for Partial Exchange Reaction of [14C]ADP into ATP with Biotin-dependent Carboxylases Scheme 7.28 (reaction is reversible)
Evidence for Enzyme-Bound Intermediate In the absence of pyruvate get a carboxylated enzyme Pyruvate carboxylase-catalyzed incorporation of 14C from H14CO3- into the enzyme Scheme 7.29 if pyruvate is added Carboxylated enzyme is unstable to acid (pH 4.5), but stable to base (0.033 N KOH) [14C] carboxylated enzyme in base purified by gel filtration then stabilized by CH2N2 treatment (makes methyl ester)
Isolation of N1-methoxycarbonylbiotin from the Reaction Catalyzed by Pyruvate Carboxylase Followed by Diazomethane Trapping of the N-carboxybiotin Scheme 7.30 The X in previous Scheme Isolated; X-ray crystal structure
Six Possible Mechanisms for Formation of N1-carboxybiotin 1. Figure 7.6 2. 3.
4. 5. 6. Figure 7.6 In the presence of HCO3- but absence of biotin, biotin carboxylase catalyzes hydrolysis of ATP; with HC18O3- one 18O incorporated into Pi; therefore supports formation of carboxyphosphate (mechanism 1).
Mechanism for the Formation of Carboxyphosphate in the Reaction Catalyzed by Acetyl-CoA Carboxylase carboxyphosphate Scheme 7.31
Possible Mechanisms for Transfer of CO2 from N1-carboxybiotin to Substrates Figure 7.8 Initial evidence for concerted: retention of configuration at -carbon
Evidence for Stepwise Mechanism Transcarboxylase and propionyl-CoA carboxylase-catalyzed elimination of HF from -fluoropropionyl-CoA Scheme 7.37 Double isotope fractionation test: Compare with If concerted, should show both 2H and 13C isotope effects (C-H bond broken and C-C bond made simultaneously) If stepwise, not necessarily so Also, if stepwise, 13C isotope effect could be different with and without 2H 13(V/K) for 13CH3COCOOH 1.0227 13(V/K) for 13CD3COCOOH 1.0141 (calculated value is 1.0136) therefore stepwise