Reactivity of Sulfhydryl Groups in Micelles

116

The reaction of the thiol group of papain with chloroacetamide, iodoacetamide, D-and L-2-bromopropionamides was studied. I n the acidic pH-range the reaction rate is higher than expected for an ordinary SH-group and shows a double-sigmoid pH-rate profile. I n addition to the already described pK, of 8.5, we found a pK, of 4.0. This indicates that in the pH-range where the enzyme is catalytically active the thiol group of Cys-25 interacts with some amino acid side chain, presumably with the imidazole group of the neighboring His-159. I n the light of the X-ray diffraction studies (Drenth et al., 1971), it is probable that the anomalous PKa of His-I59 is due to its interaction with Trp-177.The comparison of the pH-rate profiles of D-and L-2-bromopropionamide reactions indicates that there is no sifgnificant alteration in the geometry of the active site around pH 4, whereas a t slightly alkaline p H structural changes can be observed.2H,O has no effect on the rate constants of the alkylation reaction. This rules out the possibility of general base catalysis by the imidazole group of His-159.The experimental data indicate that the dissociated thiol group forms an ion pair with the protonated imidazole group. On the basis of the formation of thiolate-imidazolium ion pair, the mechanism of action of papain is reconsidered. It is concluded that the acylation and deacylation steps are not symmetrical processes, and they are one-encounter type reactions. The interaction between His-I59 and Trp-177 may be important in the catalysis.The pHdependence of the reaction of papain with chloroacetamide [I] and 2-iodopropionamide [2] is similar to that of an ordinary SH-compound. The second-order rate constants with the enzyme are about 10 times higher [I, 21. These investigations were restricted to the alkaline pH-range where the dissociated SH-group of papain is a very good nucleophile in a simple alkylation reaction. However, if the SH-group is activated by some amino acid side chain, this is to be expected rather in the acidic pH-range where papain shows high enzymatic activity. The formation of acyl-papain displays a bell-shaped pH-rate profile with pK, -4 and pK, B 8.5 [3]. The present study is focused on the acidic pH-range. It is found that with decreasing pH the rate of alkylation of the SH-group becomes higher by several orders of magnitude than that of a normal SH-group. This allows us to draw conclusions about the nature of the tjhiol group not only in the alkylation but also in the enzymatic catalysis .Abbreviation. Nbs,, 5,5'-dithio-bis(2-nitrobenzoic acid) (formerly abbreviated DTNB).Enzyme. Papain (EC 3.4.4.10). MATERIALS AND METHODSChemicals 5,5'-Dithio-bis(2-nitrobenzoic acid) (Nbs,) was purchased from Fluka AG and carbobenzyloxyglycine nitrophenyl ester from Calbiochem Co. Iodoacetamide and chloroacetamide were recrystallized from carbon tetrachloride and water, respectively ; D-and L-2-bromopropionamide were synthesized by Dr E. Moravscik. The enrichment of 2H20 was greater than 99O/,. Enzyme 0.3 ml of twice-cr...

Section: The Possibility Of Electrophilic Assistancementioning

confidence: 99%

On the Mode of Activation of the Catalytically Essential Sulfhydryl Group of Papain

Polgár

1973

116

“…In general, a thiol group does not undergo deprotonation when it is in a hydrophobic environment [11,12]. Therefore, we postulate that Cys341, with a fully protonated SH group, maintains hydrophobic interactions with neighboring amino‐acid residues.…”

Section: Discussionmentioning

confidence: 99%

“…In these previous studies, the importance of the hydrophobic property of Cys341 has been neglected. In general, when an SH group is fully protonated, it is able to form hydrophobic interactions with neighboring hydrophobic residues and aids in creating a hydrophobic environment [11,12]. To address this issue, we constructed six mutants by site‐directed mutagenesis, in which Val and Phe mutants are designed to maintain hydrophobicity and Gly, Ser, Asp and His mutants to provide hydrophilicity.…”

mentioning

confidence: 99%

Amphipathic property of free thiol group contributes to an increase inthe catalytic efficiency of carboxypeptidase Y

Mima

Jung

Onizuka

et al. 2002

Cys341 of carboxypeptidase Y, which constitutes one side of the solvent-accessible surface of the S1 binding pocket, was replaced with Gly, Ser, Asp, Val, Phe or His by site-directed mutagenesis. Kinetic analysis, using Cbz-dipeptide substrates, revealed that polar amino acids at the 341 position increased K(m) whereas hydrophobic amino acids in this position tended to decrease K(m). This suggests the involvement of Cys341 in the formation of the Michaelis complex in which Cys341 favors the formation of hydrophobic interactions with the P1 side chain of the substrate as well as with residues comprising the surface of the S1 binding pocket. Furthermore, C341G and C341S mutants had significantly higher k(cat) values with substrates containing the hydrophobic P1 side chain than C341V or C341F. This indicates that the nonhydrophobic property conferred by Gly or Ser gives flexibility or instability to the S1 pocket, which contributes to the increased k(cat) values of C341G or C341S. The results suggest that Cys341 may interact with His397 during catalysis. Therefore, we propose a dual role for Cys341: (a) its hydrophobicity allows it to participate in the formation of the Michaelis complex with hydrophobic substrates, where it maintains an unfavorable steric constraint in the S1 subsite; (b) its interaction with the imidazole ring of His397 contributes to the rate enhancement by stabilizing the tetrahedral intermediate in the transition state.

Section: Effect Of Replacement Of Cys341 On Kinetic Constantsmentioning

confidence: 91%

“…In these previous studies, the importance of the hydrophobic property of Cys341 has been neglected. In general, when an SH group is fully protonated, it is able to form hydrophobic interactions with neighboring hydrophobic residues and aids in creating a hydrophobic environment [11,12]. To address this issue, we constructed six mutants by site-directed mutagenesis, in which Val and Phe mutants are designed to maintain hydrophobicity and Gly, Ser, Asp and His mutants to provide hydrophilicity.…”

mentioning

confidence: 99%

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP‐utilizing enzymes

Garcia-Alles¹,

Erni²

2002

The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)-and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the 3 J H-C coupling constants between the carboxylate carbons and the vinyl protons.All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg 2+ -and Mn 2+ -activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg 2+ , but not with Mn 2+ . For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t 1/2 ¼ 0.7 min).Keywords: phosphoenolpyruvate analogues; chemical synthesis; inhibition; irreversible inhibitor; PEP-utilizing enzymes.Phosphoenolpyruvate (PEP) is a small and highly functionalized molecule that plays a central role in metabolism. It is not only important because of its high phosphate group-transfer potential (DG ¼ )61.9 kJAEmol )1 ), but also because it is a versatile C 3-synthon in C-C, C-P and C-O bond-formation reactions [1]. Representative examples of the first function are the synthesis of ATP catalysed by pyruvate kinase, and the transport with concomitant phosphorylation of carbohydrates across the bacterial membrane, mediated by the PEP:sugar phosphotransferase system (PTS) [2]. Examples of the second function are the fixation of CO 2 in plants (mediated by PEP carboxylase) [3], the generation of natural phosphonates (PEP mutase) [4], the first step in peptidoglycan cell-wall biosynthesis (catalysed by UDP-GlcNAc enolpyruvyl transferase) and the biosynthesis of aromatic amino acids (3-deoxy-D-arabinoheptulosonate-7-phosphate synthase and 5-enolpyruvylshikimate-3-phosphate synthase) [1].Because of its pivotal role in metabolism, PEP has been the subject of extensive chemical modification. Most of the pseudosubstrates or competitive inhibitors discovered so far differed from PEP by the presence of...