NIVEL DE SIGNIFICANCIA
VIII. LITERATURA CITADA
As mentioned previously (Section 2.3.3.0), the nature of the P4 N-protection affects the degree of binding to the proteinase of the Leu-Ala-Gly-aminoacetonitrile inhibitors. N-Protecting groups with a polarisable 7t-electron system in the P5-P6 positions, such as a benzyl group, tend to bind more strongly to the proteinase.
This evidence suggests that the proteinase has residues that can interact with a 7C-electron
system, such as a tryptophan or a phenylalanyine residue, 7c-stacldng between the benzyl
group and the side chain of the aminoacid residue (Fig. 2.29), would enhance the binding of
the Cbz-protected nitrile, compared with TFA- or t-Boc- protected nitriles.
a) b)
Fig. 2.28; TV-stacking interactions between a benzyl group and; a) aphenylalanyl residue and b) a ùyptophanyl residue.
Both Cbz-Leu-Ala-Gly-aminoacetonitrile (78) and t-Boc-Leu-Ala-Gly-aminoacetonitrile
(79) were reversible noncompetitive inhibitors of the adenovirus proteinase (Section 2.3.3.0)
2,4 Discussion J139
acetyl-Phe-aminoacetonitrile (188) (K i 0 .7 3 jamol and benzoyl-aminoacetonitrile
( 7 7 ) (K i 0 . 3 8 mmoi dm'^)/^^ are competitive inhibitors of papain.
Noncompetitive inhibition arises when an inhibitor binds to an enzymatic intermediate species and the free enzyme to the same extent (Section 1.2.3.1.3) and the level of inhibition varies with substrate concentration. It is therefore possible that the substrate could bind at the active site of the proteinase and the nitrile inhibitors bind to the proteinase, elsewhere,
modifying the conformation of the proteinase and reducing the rate of proteolytic hydrolysis.
Alternatively the inhibitor could bind at the active site of a proteinase-substrate intermediate
as well as the free enzyme.
From the series of TFA-protected nitriles (Table 2.6, Section 2 . 3 .3 . 0 ) , the nitrile inhibitors
show the same residue sequence specificity as the natural substrates. The removal of the P4
leucyl residue results in an 8 0 % loss of initial-rate-percentage-inhibition, and the loss of the
P3 alanyl residue results in the 1 0 0 % loss of initial-rate-percentage-inhibition. This implies
that the nitrile inhibitors bind in the P side substrate binding pocket, as the probability of another region of the proteinase requiring the same sequence specificity as the active site is extremely low.
Cbz-Leu-Ala-Gly-aminoacetonitrile (78) and t-Boc-Leu-Ala-Gly-aminoacetonitrile (79)
do not contain any residues in the P' positions. It may be possible for the nitriles to mimic the
P side acid leaving group, after the cleavage of the Pi-Pf amide bond. It seems likely that the nitriles can bind to the free enzyme, mimicing the substrate and bind to the proteinase after the P side acid group has left while the P’ side amine product is still attached to the proteinase. The nitriles would be binding at the active site of the proteinase and behave as a noncompetitive inhibitor which fits the observed results.
From this it is clear that the order of product release and the rate determining step differs between papain and the adenovirus type 2 proteinase. Papain initially cleaves the Pi-Pf
2.4 Discussion 140
amide bond by the formation of a thioester and the P ’ side amine product (step i. Scheme
2.48). The amine product diffuses away from the the active site of papain (step ii, Scheme 2.48) and the thioester is hydrolysed by water to give the His-Cys ion pair and the P side acid
product (step iii, Scheme 2.48). In papain cleavage of the carbon-nitrogen bond is the rate
determining step.^^^ The P side acid product then diffuses away from the active site of papain,
leaving papain free for fiuther catalytic cycles (step iv. Scheme 2.48).
P side mimics, such as acetyl-Phe-aminoacetonitrile (188) and benzoyl-aminoacetonitrile
(77) are only able to inhibit the free enzyme, as at all other steps during the catalytic cycle
the S side of the proteinase is blocked by the P side of the substrate.
-NR'Hc
+ H20 / n
R ^ oh - RCOgH
Scheme 2.48: The mechanism o f papain.
The noncompetitive nature of the nitriles suggests that the adenovirus proteinase behaves differently, as the hydrolysis products leave in a different order compared to papain. Initially the adenovirus proteinase cleaves the Pi-Pf amide bond by the formation of a thioester and the P ’ side amine product (step i. Scheme 2.49). However the amine does not leave the
2.4 Discussion 141
active site of the proteinase and the thioester is hydrolysed by water to give the His-Cys ion
pair and the P side acid product (step ii, Scheme 2.49). The P side acid product then diffuses away from the active site of papain, leaving the P ’ side amine still attached to the proteinase (step iii. Scheme 2.49). The P’ side amine product then diffuses away from the active site of the proteinase, leaving the adenovirus proteinase free of further catalytic cycles (step iv).
For the adenovirus proteinase, inhibited by the nitriles, the loss of the P ’ amine product must be the rate determining step in the hydrolysis reaction, otherwise an inhibitor binding to the enzyme-P' amine complex would have no effect on the overall reaction rate. The lack of
hydrolysis of the simple amides (Section 2.4.0) has shown that if the carbon-nitrogen bond is
planar and rotation of the carbon-nitrogen bond is restricted, additional interactions between
the P’ side of the substrate and the proteinase are required for the potential substrate to bind to the proteinase. As the cyanides contain no P’ residues it is reasonable to suppose that the nitrile functionality is able to adopt the amide binding conformation without the need for P’ interaction.
+ H2O
- RCO2H -NR'Hc
2,4 Discussion 142 The mode of binding of Cbz-Leu-Ala-Gly-aminoacetonitrile (78) and t-Boc-Leu-Ala-Gly-
aminoacetonitrile (79) is also rather intriguing. The nitrile binds to free papain allowing the
thiolate nucleophile to attack the nitrile carbon. The sp hydridised nitrogen becomes an sp^
hybridised thioamidate nitrogen and moves up into the Pi carbonyl oxygen pocket (Scheme 2.50). The Pi carbonyl oxygen binding pocket contains hydrogen bond donors, which normally polarise the Pi carbonyl bond. In this case the protons also act to stabilise the thioamidate adduct between papain and the nitrile inhibitor (Scheme 2.50). The formation of the adduct is reversible and deprotonation of the thioamidate nitrogen results in the reformation of the nitrile inhibitor and papain again.
(
carbonyl binding pocket Nitrogen binding pocketV 11
carbonyl binding pocket Nitrogen binding pocketScheme 2.50: The formation o f a thioamidate adduct between papain and a nitrile inhibitor.
In the case of the adenovirus proteinase, hydrolysis of the t-butyl ester (Section 2.4.3) and the t-butyl urethane (Section 2,4.4) along with the nature of inhibition by the epoxides (Section 2.4.5), alcohol and the bromide (Section 2.4.7), all suggest that there is no significant protonation of the carbonyl oxygen during proteolytic hydrolysis by the adenovirus proteinase. When the nitrile inhibitor binds to the adenovirus proteinase-amine
complex, the nitrile carbon can again mimic the carbonyl carbon of the amide substrate and is
attacked by the thiolate nucleophile. However when the nitrogen moves up into the Pi carbonyl oxygen binding pocket, there is no significant protonation, so a thioamidate anion
2.4 Discussion 143
(190) is formed (Scheme 2.51). The thioamidate anion (190) is not as stable as a thioamidate
and would tend to expel the thiolate nucleophile, reforming the nitrile.
carbonyl binding pocket pocket S carbonyl binding N " \ pocket I
II
R S u Nitrogen I v V * binding (1 9 0 ) ^ pocketScheme 2.51: The formation o f a thioamidate adduct (190) between the adenovirus proteinase and a nitrile inhibitor.
As no significant Pi carbonyl oxygen protonation appears to occur during the proteolytic
hydrolysis mechanism of the adenovirus proteinase, the nitrile would be a less potent inhibitor than for papain where significant protonation of the carbonyl oxygen occurs.
However direct comparison of the Kj values of inhibitors for the adenovirus proteinase and papain is not possible, as the inhibitors also have different peptide sequences. There are two effects on the affinity of the proteinase for the inhibitor: One is due to the cyanide
functionality and the other, to the peptidyl sequences. These effects can not be separated and
direct comparison of the Kfs for inhibitors is not usefial.
Retention of the P’ amine product in the proteinase’s active site is enhanced by the
formation of a partial bond between the thioamidate group’s carbon and the P ’ amine’s nitrogen (Fig. 2.29a). In this case, the thioamidate (190)-P’ amine complex (191) could
mimic the substrate (Fig. 2.29c) or the transition state complex where the carbon-nitrogen
bond of the amide is breaking while the thiolate-carbony! carbon and the nitrogen-proton bonds are forming (192) (Fig. 2.29b).
2.4 Discussion 144 carbonyl carbonyl binding binding p ocket pocket a) b) 5' 5 " S S " 6'S pocket I I (191) (192) carbonyl binding pocket c) \ • Nitrogen g- S binding s p o d tet t (193) Fig. 2.29:
a) The possible bonding interactions o f the thioamidate anion (190) and the P ’ side amine; b) the substrate transition state and c) substrate.
This leads to the possibility that the proteinase could catalyse the formation of the amide
analogues (194) and (195) by the reverse reaction of amide bond hydrolysis (Scheme 2.52).
After debinding from the proteinase the amide analogue (195) would be hydrolysed to give the simple amide (19) and the P’ amine product. The nitrile has effectively been hydrated to an amide.
Hydration of the nitrile by the proteinase could be confirmed by the loss of inhibition with time as the simple amide (19) did not inhibit the proteinase, and by the presence of the amide (19) in the hydrolysis mixture.
2,4 Discussion 145 , „ c ï ( 1 9 1 ) (1 9 4 ) . 0 / A H H H +1, H H"" H
V "
— X
c
I H H (1 9 )Scheme 2.52: The possible formation o f an amide from a nitrile by the proteinase.