1. CHEE 323J.S. Parent1 Enzymatic Synthesis of Aspartame Aspartame is a low-calorie sweetener whose apparent sweetness is 150- 200 times that of sucrose. It is prepared by condensation of L-aspartic acid and the methyl ester of L-phenylalanine (two amino acids). Its sweet taste depends on:  L-conformation of the two constituent amino acids  presence of the methyl ester  correct coupling of the amino acids. SweetBitter

2. CHEE 323J.S. Parent2 Industrial Enzymatic Synthesis of Aspartame The unique regio and stereoselectivity afforded by enzymes has been exploited on an industrial scale Aspartame production. The process employs a protease, thermolysin, to catalyze the condensation of the modified Asp and Phe). The forward reaction is written as: Note however, that the synthesis reaction is equilibrium limited by the reverse (hydrolysis) reaction for which proteases are known. Furthermore, the equilibrium strongly favours hydrolysis.

3. CHEE 323J.S. Parent3 Structural Properties of Thermolysin Thermolysin is a metalloenzyme (316 amino acids) requiring a zinc ion and four calcium ions to maintain an active tertiary structure. Two distinct hemispheres exist with a zinc atom located at the bottom of the cleft. Three residues (142, 146 and 166) serve as ligands for zinc. Calcium is a structural element, and is not believed to interact with the substrate at the active site. Open circles:  -carbon positions Stippled circle: zinc with its three protein ligands as broken lines Solid circles: four calcium atoms

4. CHEE 323J.S. Parent4 Chemical Properties of Thermolysin Thermolysin is an extracellular enzyme produced by a bacterial strain that can withstand high temperatures. Hence, themolysin has a temperature stability that is superiour to most enzymes. Thermolysin is classified as a protease, in that it catalyzes the cleavage of the peptide bonds that constitute proteins.  The term endopeptidase applies, as the internal bonds in polypeptides are susceptible to the action of thermolysin  The term neutral protease applies, as the pH optimum lies about pH 7.5  The term metalloenzyme is appropriate, given the necessity of zinc at the active site and the requirement for calcium to maintain an active tertiary structure. Chelating agents deactivate thermolysin. Enzymes of this class demonstrate substrate specificity which requires a hydrophobic amino acid such as phenylalanine as the residue whose amido group is cleaved.

5. CHEE 323J.S. Parent5 Kinetics of the Aspartame Synthesis The rate of APM production is first- order with respect to the total concentration of enzyme [E o ], and a bell-shaped pH-rate profile with the highest activity at pH 7.5 is observed. Shown is a t ypical time course of the thermolysin catalysed condensation of N-benzyloxycarbonylaspartic acid with phenylalanine methyl ester. Initial rate measurements (from t=0 to t=10 min) as a function of reagent concentrations define the overall reaction kinetics. [Z-L-Asp] =1.82 x 10 -2 M [L-PM] = 3.64 x 10 -2 M [E o ] = 4.85 x I0 -6 M pH = 6.5;  0.364 M T = 40C

6. CHEE 323J.S. Parent6 Influence of [PM] on the Condensation Rate APM synthesis is first-order WRT phenylalananine methyl ester, with no apparent saturation behaviour that is common in enzyme-mediated reactions. Note that the presence of D-PM has no effect on the reaction rate, and it is not found in the product. * [L-PM] =1.82x10 -2 M with [D-PM] =9.09x10 -3 M ** [L-PM ] =3.64 x10 -2 M with [D-PM] =1.82 x10 -2 M [Z-L-Asp] =1.82 x 10 -2 M [E o ] = 4.85 x I0 -6 M pH 6.5;  0.364 M; 40C L-PM D,L-PM

7. CHEE 323J.S. Parent7 Influence of [Z-L-Asp] on the Condensation Rate A plot of [Z-L-Asp] against the APM production rate shows saturation of the rate, typical Michaelis-Menten behaviour. Rate retardation occurs in the presence of Z-D-Asp, indicating that the enantiomer acts as a competitive inhibitor. Hence only pure L-Asp can be used in APM synthesis, while racemic mixtures of D,L-PM can be accommodated. [L-PM] =3.64 x 10 -2 M [E o ] = 4.85 x I0 -6 M pH 6.5;  0.364 M; 40C Pure Z-L-Asp 9.1x10 -3 M Z-D-Asp added

8. CHEE 323J.S. Parent8 Proposed Reaction Mechanism Competitive inhibitors reduce the rate of product formation through binding the enzyme in an inactive form.  Often these inhibitors are structurally similar to the substrate, and therefore are capable of binding at the active site  Enzyme-bound inhibitor either lacks a needed functional group or is held in an unsuitable position for reaction. We have seen an example of this behaviour in aspartame production, where the enantiomer of L-Asp inhibited the reaction. A plausible mechanism for this inhibition is shown below: Note that Z-D-Asp binds thermolysin in an inactive state, thereby reducing the active enzyme concentration and lowering the reaction rate.

9. CHEE 323J.S. Parent9 Competitive Inhibition by Z-D-Asp From this proposed mechanism we can derive a rate expression that accounts for competitive inhibition. r 1 : r 3 : r 2 : Assigning r 2 as the rate determining step of the process, we find the reaction velocity is:

10. CHEE 323J.S. Parent10 Validating the Proposed Reaction Scheme Although more sophisticated regression techniques are available, the simplest means of testing the model is to linearize the rate expression by inverting it: A plot of 1/rate versus 1/[Z-L-Asp] should be linear, with a slope of K 1 (1+[Z-DAsp]/K 3 )/(k 2 [E] T [L-PM]) and an intercept 1/(k 2 [E] T [L-PM])  This is commonly referred to as a Lineweaver-Burk plot  It is necessary that the data fit the rate expression, but it is not sufficient proof that the mechanism is correct  From the slope, intercept, [E] T and [L-PM], numerical estimates of K 1 and k 2 can be derived.

11. CHEE 323J.S. Parent11 Lineweaver-Burk Plot of the Kinetic Data Plotting the inverse of the APM production rate (moleL -1 s -1 ) against 1/[Z-L-Asp] reveals a linear relationship The proposed mechanism is consistent with the kinetic data, and may be correct.  From the slopes and intercepts, k 2 = 2.65 L mole -1 s -1 K 1 = 1.03x10 -2 mole L -1 K 3 = 2.35x10 -2 mole L -1 Line A: no Z-D-Asp; Line B: [Z-D-Asp]=9.09x10 -3 M [L-PM] = 1.82 x 10 -2 M [E o ] = 4.85 x 10 -6 M pH 6.5;  0.364 M; 40C

12. CHEE 323J.S. Parent12 Isolation of the Aspartame Product Proteases are recognized as catalysts for peptide bond cleavage, and using them to catalyze the reverse condensation reaction can be problematic. The equilibrium constant derived from the Gibbs energies of the reaction components is quite small, making the conversion of a standard batch reaction equilibrium limited.

13. CHEE 323J.S. Parent13 Isolation of the Aspartame Product Luckily APM forms, via its free side-chain carboxylic acid, a sparingly soluble addition compound with excess PM.  The synthesis can be driven using LeChatalier’s Principle by removal of the precipitation of the product.  Once isolated from the enzyme, hydrolysis of Z-APM is no longer a concern, excess PM can be removed and the product can be deprotected to yield aspartame.