BIOCHEMISTRY - DR. JAKUBOWSKI
Learning Goals/Objectives for Chapter 7B: After class and this reading, students will be able to
Chymotrypsin, a protease, cleaves amides as well as small ester substrates after aromatic residues. The following data using different chymotrypsin substrates suggests a covalent intermediate occurs on chymotrypsin catalyzed cleavage of esters and amides.
|Chymotrypsin substrate cleavage, 25oC, pH 7.9|
|kinetic constants||Acetyl-Tyr-Gly-amide||Acetyl-Tyr-O Ethylester||Ester/Amide|
Kinetic constants for chymotrypsin cleavage of N-acetyl-L-Trp Derivatives - N-acetyl-L-Trp-X
|X||kcat (s-1)||Km x 103 (M)|
We have seen a kinetic mechanism consistent with these ideas before. The reaction equations are shown below:
In this reaction, a substrate S might interact with E to form a complex, which then is cleaved to products P and Q. Q is released from the enzyme, but P might stay covalently attached, until it is expelled. This conforms exactly to the mechanism described above. For chymotrypsin-catalyzed cleavage, the step characterized by k2 is the acylation step (with release of the leaving group such as p-nitrophenol in Lab 5). The step characterized by k3 is the deacylation step in which water attacks the acyl enzyme to release product P (free phosphate in Lab 5). In class and for homework you derived the following equation::
Equation 7: v = [(k2k3)/(k2 + k3)]EoS/[Ks(k3)/(k2+k3)] + S
As mentioned above, for hydrolysis of ester substrates, which have better leaving groups compared to amides, deacylation is rate limiting, ( k3<<k2). Then equation 7 becomes
v = k3EoS/[Ks(k3)/(k2) + S]
Vm = k3Eo and Km = Ks(k3)/(k2)
For amide hydrolysis, as mentioned above, acylation can be rate-limiting (k2<<k3). Then equation 7 becomes:
v = k2EoS/[Ks+ S]
Vm = k2Eo and Km = Ks
Just as we saw before for the rapid equilibrium assumption (when ES falls apart to E + S more quickly than it goes to product), Km = Ks in amide hydrolysis.
Note: A new theoretical computer program,
THEMATICS (theoretical microscopic titration curves) has been developed
to calculate the titration curves for all ionizable groups in a protein.
When performed on test proteins, those amino acids that showed anomalous
curves (flattened compared to normal titration curves) where usually found
in the active site of the protein. The flattened curves show that the
amino acid is partially protonated over a wider range of pH then
theoretically expected. The program can be used to predict active site
regions on protein of known structure but unknown function, and will be
useful in the emerging field of proteomics. (figure below from Proc. Natl.
Acad. Sci. USA, Vol. 98, Issue 22, 12473-12478, October 23, 2001)
Fig. 1. Sample theoretical titration curves. Predicted mean net charge as a function of pH. (A) All of the histidine residues in the A chain of TIM: His-26 (+), His-95 (ï¿½), His-100 (*), His-115 (open square), His-185 (filled square), His-195 (open circle), His-224 (filled circle), and His-248 (). (B) Selected tyrosine residues of AR: Tyr-39 (+), Tyr-48 (ï¿½), Tyr-177 (*), Tyr-291 (open square), and Tyr-309 (filled square). (C) Selected lysine residues of PMI: Lys-100 (+), Lys-117 (ï¿½), Lys-128 (*), Lys-136 (open square), and Lys-153 (filled square). TIM, triosephosphate isomerase; AR, aldose reductase; PMI, phosphomannose isomerase. Remember that depending on the protein microenvironment, the pKa of a side chains like Asp can vary from 0.5 to 9.2!
Malathion and ethyl parathion (organic phosphate pesticides) have similar structures and reactivities as DIPF, and selectively inhibit serine proteases in insects.
Figure: tos-L-Phe-chloromethyl ketone
Figure: Serine Protease Mechanism
In short, all the catalytic mechanisms we encountered previously are at play in chymotrypsin catalysis. These include nucleophilic catalysis (with the Ser 195 forming a covalent intermediate with the substrates), general acid/base catalysis with His 57, and loosely, electrostatic catalysis with Asp 102 stabilizing not the transition state or intermediate, but the protonated form of His 57. An important point to note is that His, as a general acid and base catalyst, not only stabilizes developing charges in the transition state, but also provides a path for proton transfer, without which reactions would have difficulty in proceeding. One final mechanism is at work. The enzyme does indeed bind the transition state more tightly than the substrate. Crystal structures with poor "pseudo"-substrates that get trapped as partial tetrahedrally-distorted substrates of the enzyme and with inhibitors show that the oxyanion intermediate, and hence presumably the TS, can form H-bonds with the amide H (from the main chain) of Gly 193 and Ser 195. These can not be made to the trigonal, sp2 hybridized substrate. In the enzyme alone, the hole into which the oxyanion intermediate and TS would be placed is not occupied. This oxyanion hole is occupied in the tetrahedral intermediate.
Many enzymes have active site serines which act as nucleophilic catalysts in nucleophilic substitution reactions (usually hydrolysis). One such enzyme is acetylcholine esterase which cleaves the neurotransmitter acetylcholine in the synapse of the neuromuscular junction. The transmitter leads to muscle contraction when it binds its receptor on the muscle cell surface. The transmitter must not reside too long in the synapse, otherwise muscle contraction will continue in an uncontrolled fashion. To prevent this, a hydrolytic enzyme, acetylcholine esterase, a serine esterase found in the synapse, cleaves the transmitter, at rates close to diffusion controlled. Diisopropylphosphofluoridate (DIPF) also inhibits this enzyme which effectively makes it a potent chemical warfare agent. An even more fluoride-based inhibitor of this enzyme, sarin, is the most potent lethal chemical agent of this class known. Only 1 mg is necessary to kill a human being.
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