Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase

Figures & data

Figure 1.  GGT reactions with γ-Glutamyl Substrates. Cleavage of glutathione, a physiologic substrate of GGT, is shown. The γ-glutamyl bond between the γ-carbon of glutamate and the amine of cysteine is cleaved by GGT. The γ-carbon of glutamate forms an acyl covalent bond with the hydroxyl group on the side chain of Thr-381 of human GGT. The acyl bond can either be hydrolyzed releasing glutamate (Glu), or an acceptor nucleophile can attack forming a new γ-glutamyl compound (transpeptidation reaction). The reactions proceeds via a Ping-Pong mechanism.

Figure 2.  Synthesis scheme for the OU749 analogs. Synthesis and purification of the synthetic intermediates TDA1-10 (A) was required before synthesis of the OU749 analogs Compounds 2–4 and Compounds 6–20 (B). Synthesis of Compound 5 is derived from Compound 11 (C).

Table 1.  Kinetic parameters for reaction with d- and l-GpNA.

Kinetic parameter	d-GpNA minus acceptor	d-GpNA plus acceptor	l-GpNA minus acceptor	l-GpNA plus acceptor
V (mM/min/nM)	2.1 ± 0.2	53 ± 11	6.5 ± 0.2	141 ± 11
V/Kglygly (min^−1nM^−1)		7.6 ± 1.5		14 ± 1
V/KGpNA (min^−1nM^−1)	13.2 ± 0.1	12.7 ± 2.5	7.8 ± 0.2	117 ± 9
Kglygly (mM)		7.0 ± 0.3		9.9 ± 0.9
KGpNA (mM)	0.16 ± 0.02	4.2 ± 0.1	0.83 ± 0.06	1.2 ± 0.1

^aThe acceptor is glygly.

Figure 3.  Initial velocity pattern with d-GpNA or l-GpNA as the substrate and glygly as an accepter. Initial rates were measured at pH 7.4 and 37 °C. Double recipricol plots of the initial velocity versus the glygly concentration with 0.25 mM (♦),0.5 mM (•),1 mM (▴), or 3 mM (▪) d-GpNA (A) or l-GpNA (B).

Table 2.  Inhibition of GGT by structural analogs of OU749.

Compound #	Substitution	Hydrolysis d-GpNA Kii (µM)	Transpeptidation l-GpNA Kii (µM)	Transpeptidation glygly Kis(µM)
OU749	None	73.2 ± 8.9	67.6 ± 6.3	54.1 ± 3.9
2	X = CH3	73.3 ± 4.0	22.7 ± 0.5	10.0 ± 0.4
3	W = Cl	74.9 ± 2.2	22.0 ± 0.7	11.9 ± 1.1
4	X = Cl; V = Cl	75.6 ± 7.2	15.7 ± 0.7	3.7 ± 1.0
5	X= NHOH	149.4 ± 13.6	64.4 ± 5.1	25.7 ± 1.3
6	WV = Benzene	161.0 ± 4.5	74.9 ± 3.6	45.7 ± 3.4
7	X = C(CH3)3	341.5 ± 6.4	149.9 ± 13.0	143.2 ± 8.7
8	X = Cl; W = Cl	3160 ± 90	18.2 ± 0.7	10.8 ± 0.8
9	X = OCH3	3800 ± 720	149 ± 23	133.3 ± 10.3
10	X = Cl	5530 ± 1460	32.5 ± 0.9	13.4 ± 0.8
11	X = NO2	Activator	92.8 ± 4.3	95.7 ± 7.6
12	X = CF3	Activator	116.7 ± 7.4	46.3 ± 3.5

Figure 4.  Kinetic analysis of GGT inhibition by OU749 and SBS. The structure of OU749 (A). Double-reciprocal plot of the initial velocity of the hydrolysis of d-GpNA in the presence of 0 (♦), 15.2 µM (▾), 31.25 µM (▴), 62.5 µM (▪), 125 µM (•) OU749 (B). The structure of SBS (C). Double reciprocal plot of the initial velocity of the hydrolysis of d-GpNA in the presence of 0 (♦), 6.25 mM (▾), 12.5 mM (▴), 25 mM (▪), 50 mM (•) SBS (D). Double reciprocal plot of the initial velocity of the transpeptidation reaction with varying concentrations of l-GpNA and 40 mM glygly (E) or varying concentrations of glygly with 3 mM l-GpNA (F) in the presence of 0 (♦), 6.25 mM (▾), 12.5 mM (▴), 25 mM (▪), 50 mM (•) SBS. Data shown are average triplicate values ± S.D. For many data points the S.D. is smaller than the symbol.

Figure 5.  Kinetic analysis of GGT inhibition by Compound 11. Substrate velocity curve of the hydrolysis reaction versus d-GpNA concentration in the presence of 0 (♦), 15.2 µM (▾), 31.25 µM (▴), 62.5 µM (▪), 125 µM (•) Compound 11 (A). Velocity vs Compound 11 concentration (closed symbols) and V/K vs Compound 11 concentration (open symbols) plots illustrate the activation of the hydrolysis reaction (B). Double-reciprocal plots of the initial velocities of the transpeptidation reaction varying l-GpNA with 40 mM glygly (C) or varying glygly with 3 mM l-GpNA (D) in the presence of 0 (♦), 15.2 µM (▾), 31.25 µM (▴), 62.5 µM (▪), 125 µM (•) Compound 11. Data shown are average triplicate values ± S.D.

Table 3.  Kinetic parameters for activators of the D-GpNA hydrolysis reaction and Inhibition of the L-GpNA transpeptidation reaction.

Compound #	Substitution	Hydrolysis D-GpNA				Inhibition L-GpNA
		Vo^a (mM/min/nM)	V∞ (mM/min/nM)	KAct (µM)	Fold activation	Transpeptidation l-GpNA Kii (µM)	Transpeptidation glygly Kis (µM)
13	None	1.59 ± 0.06	9.09 ± 0.55	54.4 ± 3.0	5.72 ± 0.32	111.5 ± 5.5	59.6 ± 3.7
14	Y = NO2	1.27 ± 0.01	5.08 ± 0.41	51.9 ± 0.8	4.00 ± 0.11	132.5 ± 5.5	81.2 ± 8.8
15	Z = Cl	1.30 ± 0.01	5.86 ± 0.23	38.6 ± 0.3	4.51 ± 0.05	119.1 ± 7.1	43.8 ± 7.7
11^b	Y = OCH3	1.59 ± 0.14	8.63 ± 1.21	37.1 ± 5.6	5.43 ± 2.04	92.8 ± 4.3	95.7 ± 7.6
16	Y = Cl	1.71 ± 0.09	7.70 ± 0.46	31.4 ± 2.2	4.50 ± 0.63	61.9 ± 2.4	44.4 ± 3.4
17	Y = CH3	2.62 ± 0.79	11.25 ± 4.27	25.3 ± 7.6	4.29 ± 2.20	112.5 ± 5.2	56.9 ± 8.3
18	Z = CH3	1.15 ± 0.01	5.08 ± 0.20	25.2 ± 0.2	4.42 ± 0.64	121.5 ± 8.8	34.3 ± 0.9
19	Y = F	1.53 ± 0.09	5.39 ± 0.28	22.2 ± 2.0	3.52 ± 0.60	Time Dependent	31.8 ± 0.7
20^c	Y = Cl Z = Cl	—	—	—	—	Time Dependent	10.3 ± 0.5

^aV_o = maximum velocity at zero activator; V_∞ = V_o(K_ID/K_IN) in eq. 2, maximum rate at infinite activator; K_act, activation constant or concentration that gives ½(V_∞ + V_o), K_ID in eq. 2; fold activation = V_∞/V_o.

^bNormalization of all values to Compound 11 yielded values similar to Compound 11.

^cCompound 20 competitively inhibited the hydrolysis reaction with a K_ii with d-GpNA of 78.2 ± 1.9 µM.

Figure 6.  Time courses for the hydrolysis of d-GpNA in the presence of acivicin. The release of pNA from 0.287 mM d-GpNA (A) or 2 mM d-GpNA (B) was monitored over time in the presence of acivicin (concentrations of acivicin noted in A). The reactions were conducted at pH 7.4. The increase in the Km of d-GpNA with increasing concentrations of acivicin is shown (insert 6A).

Figure 7.  Kinetic analysis of GGT inhibition by Compound 20. Double-reciprocal plot of the initial velocity of the hydrolysis of d-GpNA in the presence of 0 (♦), 15.2 µM (▾), 31.25 µM (▴), 62.5 µM (▪), 125 µM (•) Compound 20 (A). Double reciprocal plot of the initial velocity of the transpeptidation reaction with varying concentrations of glygly with 3 mM l-GpNA (B) in the presence of 0 (○), 15.2 µM (♦), 31.25 µM (▾), 62.5 µM (▴), 125 µM (▪), 250 µM (•) Compound 20. Data shown are average triplicate values ± S.D. For many data points the S.D. is smaller than the symbol.

Figure 8.  Proposed overall kinetic mechanism of inhibition and activation of GGT. Mechanism of hydrolysis of d-GpNA (A). Enzyme (E) is γ-glutamylated by d-GpNA with release of pNA to give a form of the enzyme (F) that can transfer the γ-glutamyl moiety to water. d-GpNA is not capable of acting as an acceptor. The ester bond can be hydrolyzed with rate constant k_hyd, and this rate is decreased in the presence of an inhibitor such as OU749 (F-I), or increased in the presence of an activator such as Compound 11 giving rate k’_hyd. Acivicin can bind to the free enzyme at the acceptor site, and is slowly trapped (tightly bound) on enzyme. Mechanism of transpeptidation of GpNA (B). Enzyme (E) is γ-glutamylated by GpNA with release of pNA to give a form of the enzyme (F) that can transfer the γ-glutamyl moiety to an acceptor (l-GpNA and/or glygly). The ester bond can be hydrolyzed with rate constant k_hyd, but competition between water, l-GpNA and/or glygly decreases the rate of hydrolysis. As in panel A, acivicin can bind to the free enzyme at the acceptor site, and is slowly trapped on enzyme.