PMID- 18366187
OWN - NLM
STAT- MEDLINE
DCOM- 20080610
LR  - 20131121
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 47
IP  - 16
DP  - 2008 Apr 22
TI  - The electrostatic driving force for nucleophilic catalysis in L-arginine 
      deiminase: a combined experimental and theoretical study.
PG  - 4721-32
LID - 10.1021/bi7023496 [doi]
AB  - L-arginine deiminase (ADI) catalyzes the hydrolysis of L-arginine to form 
      L-citrulline and ammonia via two partial reactions. A working model of the ADI 
      catalytic mechanism assumes nucleophilic catalysis by a stringently conserved 
      active site Cys and general acid-general base catalysis by a stringently 
      conserved active site His. Accordingly, in the first partial reaction, the Cys 
      attacks the substrate guanidino C zeta atom to form a tetrahedral covalent 
      adduct, which is protonated by the His at the departing ammonia group to 
      facilitate the formation of the Cys- S-alkylthiouronium intermediate. In the 
      second partial reaction, the His activates a water molecule for nucleophilic 
      addition at the thiouronium C zeta atom to form the second tetrahedral 
      intermediate, which eliminates the Cys in formation of the L-citrulline product. 
      The absence of a basic residue near the Cys thiol suggested that the 
      electrostatic environment of the Cys thiol, in the enzyme-substrate complex, 
      stabilizes the Cys thiolate anion. The studies described in this paper explore 
      the mechanism of stabilization of the Cys thiolate. First, the log(k(cat)/K(m)) 
      and log k(cat) pH rate profiles were measured for several structurally divergent 
      ADIs to establish the pH range for ADI catalysis. All ADIs were optimally active 
      at pH 5, which suggested that the Cys pKa is strongly perturbed by the prevailing 
      electrostatics of the ADI active site. The p K a of the Bacillus cereus ADI 
      (BcADI) was determined by UV-pH titration to be 9.6. In contrast, the pKa 
      determined by iodoacetamide Cys alkylation is 6.9. These results suggest that the 
      negative electrostatic field from the two opposing Asp carboxylates perturbs the 
      Cys pKa upward in the apoenzyme and that the binding of the iodoacetamide (a 
      truncated analogue of the citrulline product) between the Cys thiol and the two 
      Asp carboxylates shields the Cys thiol, thereby reducing its pKa. It is 
      hypothesized that the bound positively charged guanidinium group of the 
      L-arginine substrate further stabilizes the Cys thiolate. The so-called 
      "substrate-assisted" Cys ionization, first reported by Fast and co-workers to 
      operate in the related enzyme dimethylarginine dimethylaminohydrolase [Stone, E. 
      M., Costello, A. L., Tierney, D. L., and Fast, W. (2006) Biochemistry 45, 
      5618-5630], was further explored computationally in ADI by using an ab initio 
      quantum mechanics/molecular mechanics method. The energy profiles for formation 
      of the tetrahedral intermediate in the first partial reaction were calculated for 
      three different reaction scenarios. From these results, we conclude that 
      catalytic turnover commences from the active configuration of the ADI(L-arginine) 
      complex which consists of the Cys thiolate (nucleophile) and His imidazolium ion 
      (general acid) and that the energy barriers for the nucleophilic addition of Cys 
      thiolate to the thiouronium C zeta atom and His imidazolium ion-assisted 
      elimination from the tetrahedral intermediate are small.
FAU - Li, Ling
AU  - Li L
AD  - Department of Chemistry and Chemical Biology, University of New Mexico, 
      Albuquerque, NM 87131, USA.
FAU - Li, Zhimin
AU  - Li Z
FAU - Wang, Canhui
AU  - Wang C
FAU - Xu, Dingguo
AU  - Xu D
FAU - Mariano, Patrick S
AU  - Mariano PS
FAU - Guo, Hua
AU  - Guo H
FAU - Dunaway-Mariano, Debra
AU  - Dunaway-Mariano D
LA  - eng
GR  - AI 059733/AI/NIAID NIH HHS/United States
GR  - R03 AI 068672/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20080327
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Sulfhydryl Compounds)
RN  - EC 3.- (Hydrolases)
RN  - EC 3.5.3.6 (arginine deiminase)
RN  - K848JZ4886 (Cysteine)
SB  - IM
MH  - Binding Sites
MH  - Catalysis
MH  - Cysteine/chemistry
MH  - Enzyme Activation/drug effects
MH  - Enzyme Inhibitors/pharmacology
MH  - Hydrogen-Ion Concentration
MH  - Hydrolases/antagonists & inhibitors/*chemistry/genetics/*metabolism
MH  - Hydrolysis
MH  - Kinetics
MH  - Models, Molecular
MH  - Molecular Structure
MH  - Pseudomonas aeruginosa/enzymology/genetics
MH  - Spectrophotometry
MH  - Static Electricity
MH  - Sulfhydryl Compounds/chemistry
EDAT- 2008/03/28 09:00
MHDA- 2008/06/11 09:00
CRDT- 2008/03/28 09:00
PHST- 2008/03/28 09:00 [pubmed]
PHST- 2008/06/11 09:00 [medline]
PHST- 2008/03/28 09:00 [entrez]
AID - 10.1021/bi7023496 [doi]
PST - ppublish
SO  - Biochemistry. 2008 Apr 22;47(16):4721-32. doi: 10.1021/bi7023496. Epub 2008 Mar 
      27.