scholarly journals Chemical mechanism of the endogenous argininosuccinate lyase activity of duck lens δ2-crystallin

1998 ◽  
Vol 333 (2) ◽  
pp. 327-334 ◽  
Author(s):  
Chi-Yue WU ◽  
Hwei-Jen LEE ◽  
Shih-Hsiung WU ◽  
Shui-Tein CHEN ◽  
Shyh-Horng CHIOU ◽  
...  
Keyword(s):  
Bond Cleavage ◽  
Isotope Effects ◽  
Enzymic Activity ◽  
Chemical Mechanism ◽  
Binary Complex ◽  
Argininosuccinate Lyase ◽  
Pka Values ◽  
Diethyl Pyrocarbonate ◽  
Lyase Activity ◽  
Ph Range

The endogenous argininosuccinate lyase activity of duck δ2-crystallin was specifically inactivated by the histidine-specific reagent, diethyl pyrocarbonate. The protein was protected by l-citrulline or l-arginine from the diethyl pyrocarbonate inactivation. To characterize further the chemical mechanism of the δ2-crystallin-catalysed reaction, deuterium-labelled argininosuccinate was enzymically synthesized from fumarate and l-arginine with δ2-crystallin in 2H2O. The argininosuccinate synthesized contained about 19% of the anhydride form; however, the deuterium was clearly demonstrated to be incorporated enantioselectively. Only the pro-HR atom at C-9 of the succinate moiety was labelled in the [2H]argininosuccinate-9-d synthesized, which indicates an anti-elimination mechanism for the endogenous argininosuccinate lyase activity of δ2-crystallin. The enzymic activity of duck lens δ2-crystallin in the pH range 5.5–8.5 was investigated using both protium- and deuterium-labelled argininosuccinate as the substrate. From the log kcat versus pH plot, two molecular pKa values of 6.18±0.02 and 8.75±0.03 were detected in the δ2-crystallin–argininosuccinate binary complex. The former must be dehydronated and the latter hydronated to achieve an optimum reaction rate. The log kcat/Km versus pH plot suggested two molecular pKa values of 5.96±0.09 and 8.29±0.10 for the free δ2-crystallin to be involved in the substrate binding. Small kinetic isotope effects of 1.17±0.02 and 1.05±0.09 were found for kcat and kcat/Km respectively. Combining results from labelling and kinetic analysis indicates that the endogenous argininosuccinate lyase activity of duck δ2-crystallin is compatible with a stepwise E1cB mechanism, the rate-limiting step probably at the C–N bond-cleavage step.

scholarly journals Inactivation of the endogenous argininosuccinate lyase activity of duck δ-crystallin by modification of an essential histidine residue with diethyl pyrocarbonate

1993 ◽  
Vol 293 (2) ◽  
pp. 537-544 ◽  
Author(s):  
H J Lee ◽  
S H Chiou ◽  
G G Chang
Keyword(s):  
Enzyme Activity ◽  
Rate Constant ◽  
Order Rate Constant ◽  
Histidine Residue ◽  
Argininosuccinate Lyase ◽  
First Order ◽  
Diethyl Pyrocarbonate ◽  
Order Rate ◽  
Lyase Activity ◽  
Pseudo First Order

The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.

scholarly journals Evidence that the active centre of chymopapain A is different from the active centres of some other cysteine proteinases and that the Brønsted coefficient (βnuc.) for the reactions of thiolate anions with 2,2′-dipyridyl disulphide may be decreased by reagent protonation

1980 ◽  
Vol 189 (1) ◽  
pp. 189-192 ◽  
Author(s):  
K Brocklehurst ◽  
B S Baines ◽  
M S Mushiri
Keyword(s):  
Carica Papaya ◽  
Cysteine Proteinases ◽  
Active Centre ◽  
Pka Values ◽  
Nitrobenzoic Acid ◽  
Specific Reactivity ◽  
Ph Range ◽  
Thiolate Anions ◽  
Active Centres

The active centres of chymopapains A and B (jointly designated EC 3.4.22.6) and papaya (Carica papaya L.) peptidase A were investigated by using 2,2′-dipyridyl disulphide and 5,5′-dithiobis-(2-nitrobenzoic acid) as thiol-specific reactivity probes. Whereas the first active-centre pKa values for chymopapain B and papaya peptidase A are less than 5, is as the case for papain (EC 3.4.22.2) and ficin (EC 3.4.22.3), that for chymopapain A is about 6.8. The reason why the reactions of thiols of pKa approx. 6.5 with 2.2′-dipyridyl disulphide are essentially pH-independent in the pH range around the thiol pKa is delineated. The value of the Brønsted coefficient (beta nuc.) for the reactions of thiolate ions with the 2,2′-dipyridyl disulphide monocation appears to be smaller than its value for the corresponding reactions with the neutral disulphide.

Determination of the Chemical Mechanism of Malic Enzyme by Isotope Effects†

Biochemistry ◽  
1997 ◽  
Vol 36 (5) ◽  
pp. 1141-1147 ◽  
Author(s):  
William A. Edens ◽  
Jeffrey L. Urbauer ◽  
W. W. Cleland
Keyword(s):  
Malic Enzyme ◽  
Isotope Effects ◽  
Chemical Mechanism

Carbon-13 and nitrogen-15 isotope effects as a probe of the chemical mechanism of Escherichia coli aspartate transcarbamylase

Biochemistry ◽  
1992 ◽  
Vol 31 (28) ◽  
pp. 6577-6584 ◽  
Author(s):  
Laura E. Parmentier ◽  
Paul M. Weiss ◽  
M. H. O'Leary ◽  
H. K. Schachman ◽  
W. W. Cleland
Keyword(s):  
Escherichia Coli ◽  
Isotope Effects ◽  
Chemical Mechanism ◽  
Aspartate Transcarbamylase

scholarly journals Femtosecond real-time probing of reactions. XIV. Rydberg states of methyl iodide

1994 ◽  
Vol 72 (3) ◽  
pp. 947-957 ◽  
Author(s):  
Hua Guo ◽  
Ahmed H. Zewail
Keyword(s):  
Methyl Iodide ◽  
Theoretical Calculations ◽  
Bond Cleavage ◽  
Isotope Effects ◽  
Rydberg States ◽  
Chem Phys ◽  
Two Dimensions ◽  
Special Focus ◽  
Methyl Radical ◽  
Valence States

The elementary reaction dynamics of methyl iodide in two Rydberg states leading to an iodine and a methyl radical occur on the femtosecond time scale (M.H. Janssen, M. Dantus, H. Guo, and A.H. Zewail. Chem. Phys. Lett. 214, 281 (1993)). In this article, we consider the dynamics of this elementary process which involves both the Rydberg and valence states. Direct comparisons are made between theory and experiment with special focus on the following observations: large isotope effects, mode dependence of the predissociation rates, and coherence effects. The quantal molecular dynamics in two-dimensions show that the initial wave packet motion occurs along a vibrational mode involving the light atoms accompanied by transitions from the Rydberg state to the repulsive state; subsequent dynamics on the dissociative state lead to the C—I bond cleavage. The theoretical calculations also give the decay behavior of the Rydberg states with lifetimes in agreement with those observed in the femtosecond experiments. Moreover, the large isotope effect in observed predissociation rates of CH3I and CD3I has been successfully reproduced by the same model. The two-dimensional dynamics underscore the shortcomings of a one-dimensional picture in which the C—I serves as the sole reaction coordinate. The model presented here offers a viable mechanism for the dynamics of these Rydberg states.

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