Once the experiments had been completed for the (2S)-serine O-sulphate inhibitors the information was depicted in two ways. The first was to plot the apparent first order rate constant (kapp) against the concentration of inhibitor [1] and fit to the Michaelis- Menten curve (Figure 2.4) (see section 2.6). The second was to transform the Michaelis- Menten fit to a straight line, 1/kapp against 1/[l] (Figure 2.5) (see section 2.6). Kj and kjnact can be obtained from these graphs, the intercept of the 1/[l] axis is equal to -1/K; and the intercept of the 1/kgpp axis is equal to kjnact - The values obtained for the direct non linear fit of the data using Enzfittei^^® are shown in Table 2.4:-
Table 2.4. Values of K| and kjnact for the Inactivation of GAD by (2S)- and (2S)-[2-2H]-serine O- sulphate.
Inhibitor Ki (mM) kinact (min-^)
(2S)-serine O-sulphate (2S)-[2-2h]-serine O-sulphate 2-57 ± 0.519 7.59 ± 2.225 0.01076 0.01368 ± 0.00098 ± 0.00264
The graphs obtained from the data for the inactivation of GAD by (2S)- and (2S)-[2-^H]- serine O-sulphate are shown in Figures 2.4 and 2.5.
0.010 0.008 0.006 0.004 0.002 0.000 0.001 0.002 0.003 0.004 0.005 [I] 0.006
Figure 2.4. kapp against [I] for the Inhibition of GAD by + (2S)-serine O-sulphate and X (2S)-[2-2H]-serine O-sulphate.
1000 800 600 400 200 0 1000 1/[l] 1200 0 200 400 600 800
Figure 2.5.1/kapn against 1/[l] for + {2S)-serine O-sulphate and X (2S)-[2-^H]-serine O-sulphate inhibition of GAD.
From the data in Table 2.4, it can be seen that kjnact is approximately equal for both of the inhibitors, and so at constant enzyme concentration (or VhA/q) was about 1.
However the values for Kj are significantly different and °(V/K) [or (VhA/d)/(Kh/Kd)] was 2.3, The value ^(V/K) indicates that C“-H bond cleavage does, indeed, occur during the inactivation of GAD. The large size of ^(V/K) indicates that the reaction commitments to C-H bond cleavage are not large and that C-H bond cleavage is not the most significant transition state in the inactivation process.^^^ If the C-H bond cleavage was the rate determining step then there would also be a significant isotope effect on V as Vmax would be decreased for the deuteriated inhibitor and this is clearly not the case. After the C® bond has broken, the intermediates are identical and therefore have identical reaction rates. Hence the rate determining step is prior to the first irreversible step (the C-H bond breakage). Binding to the enzyme active site and transaldimination of the inhibitor with the active-site lysine bound to the PLP have both occurred before the C-H bond breaks. It is possible that either of these two processes is rate determining.
For (2S)-serine O-sulphate we have shown that a kinetically significant step in the inactivation process is the breakage of the C“-H bond which supports the mechanism proposed by Metzler.''®® An incubation of (2S)-serine O-sulphate with GAD in 0.1 M pyridine/DCI buffer at pH 4.6 was studied by proton NMR spectroscopy. No exchange of the a-proton occurred after several days. This observation implies that the anion
formed on the loss of the a-proton is not stabilised, and almost Immediately the C-C 4 double bond is formed and HO3SO' is eliminated from the enzyme active site. Hence,
the inactivation process has been shown to occur via the removal of a proton from the C-4'-re-face of the coenzyme and the data is most consistent with an E2 type elimination reaction.
2.7.2 (2R)-Serlne O -sulphates
At all concentrations studied for (2R)-serine O-sulphate, the deuteriated (2R)-serine O- # sulphate inactivated the enzyme faster than the unlabelled isotopomer, so there is an
inverse isotope effect. The averaged apparent values rates of inactivation for the two inhibitors (Vh and Vp) and the ratio of rates {VhA/q) are shown in Table 2.5.
Table 2.5. The average rates of inactivation of GAD by (2R)- and (2R)-[2-2H]-serine O-sulphate,
Concentration (mM) Vh (X 10-3) Vd (X 10-3) Vh/Vd 2.0 0.921 1.419 0.649 3.5 1.321 2.598 0.509 5.0 1.560 3.891 0.411 7.0 1.861 5.821 0 .320
Î
a
The isotope effect is clearly inverse, but could not be determined accurately from the narrow range of inhibitor concentrations amenable to kinetic analysis, as the rate of Inactivation at points near V^ax was too fast to be measured using the radiochemical method. Nevertheless, the existence of an inverse isotope effect indicates thatC“-C0 2‘
bond cleavage occurs. The C“-C0 2‘ bond must be disposed on the 4’- re-face of the coenzyme as the binding position of the distal sulphate group is identical for all substrates and inhibitors,and so the reaction occurs on the C-4’-re-face of the coenzyme. The bond cleavage step is rate-limiting, and the observed isotope effect values could represent secondary a-isotope effects where the bond is stiffer in the transition state than in the product external aldimine.^^® However, the magnitude of the measured inverse isotope effect is too large to be due a secondary a-isotope effect
and a more likely explanation is that there are alternative reaction pathways which do 1
96
not lead to inactivation of the enzyme which are suppressed on the addition of deuterium. This would make the rate of inactivation faster for the deuteriated inhibitor than the non deuteriated one, this is an induced isotope effect. ‘^(V/K) for the inactivation reaction was 1.0 indicating that there is a large reaction commitment to the
first isotopically sensitive step and that the suicide substrate is extremely stick y,
contrast to the (2S)-enantiomer. The data is consistent with an early C-OSO3 bond
ionisation.
In order to verify our conclusion we wanted to inactivate GAD with (2R)-[U-^^G]-serine O-sulphate. This study had not been carried out previously due to the lack of availability of (2R)-[U-i'^C]-serine (section 2.4). In order to obtain a sample of (2R)-[U- ^^C]-serine O-sulphate, (2S)-[U-^^C]-serine would need to be racemised, followed by the resolution of the racemate to yield (2R)-[U-^^C]-serine. The (2R)-[U-^^C]-serine could then be converted into (2R)-[U-^^C]-serine O-sulphate. The synthesis of (2R)-[U-
97
"I. A proton NMR experiment with (2R)-serine O-sulphate (0.226 mmol) and GAD (8.467
nmoles) in 1 ml of 0.1 M pyridine/DCI buffer at pD 4.2 was carried out. No new signals
were detected for times up to 113 hours. However, the maximum amount of ‘I Schnackerz adduct produced would only be about 1% of the amount of (2R)-serine O-
sulphate present at t=0, and so would not be seen. The rate of the alternative reaction pathway cannot be slower than the inactivation or the introduction of deuterium would not result in the Increased rate of the pathway leading to inactivation. It is likely that the two rates are similar and, therefore, It is hardly surprising that no new signals were seen in the proton NMR spectrum. It was impractical to attempt to isolate the products of the inactivation reaction as a very large amount of enzyme would be required to form a realistic amount of the Schnackerz adduct.
1
^'^Cj-serine O-sulphate in this manner would be time consuming due to the resolution step. The possibility of incubating (2RS)-[U-^'^C]-serine O-sulphate (151) with the enzyme was therefore investigated. Any ^"^002 evolved would be from the (2R)-[U-^'^C]- serine O-sulphate as (2S)-[U-^'^C]-serine O-sulphate does not liberate ^"^002 during the
inactivation process (section 2.2.4).
N-Acetyl-(2RS)-serine (152) was prepared by reaction of (2S)-serine with acetic