Current problems in mechanistic studies of serine and cysteine proteinases

Polgár, László; Halász, Piroska

doi:10.1042/bj2070001

Cited by 164 publications

(73 citation statements)

References 109 publications

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“…1C). This diagnostic downfield shift provides strong evidence supporting the protonation of His-269 in the S122C mutant and formation of an imidazole-thiolate ion pair between the His-269 and Cys-122 in the modified hMGL active site (24). Gradual titration to a higher pH elicited a decrease in the peak intensity at 18 ppm, and this resonance disappeared completely at pH 10.6 (data not shown).…”

Section: Resultsmentioning

confidence: 83%

Specific Inter-residue Interactions as Determinants of Human Monoacylglycerol Lipase Catalytic Competency

Tyukhtenko

Karageorgos

Rajarshi

et al. 2016

Journal of Biological Chemistry

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The serine hydrolase monoacylglycerol lipase (MGL) functions as the main metabolizing enzyme of 2-arachidonoyl glycerol, an endocannabinoid signaling lipid whose elevation through genetic or pharmacological MGL ablation exerts therapeutic effects in various preclinical disease models. To inform structure-based MGL inhibitor design, we report the direct NMR detection of a reversible equilibrium between active and inactive states of human MGL (hMGL) that is slow on the NMR time scale and can be modulated in a controlled manner by pH, temperature, and select point mutations. Kinetic measurements revealed that hMGL substrate turnover is rate-limited across this equilibrium. We identify a network of aromatic interactions and hydrogen bonds that regulates hMGL active-inactive state interconversion. The data highlight specific inter-residue interactions within hMGL modulating the enzymes function and implicate transitions between active (open) and inactive (closed) states of the hMGL lid domain in controlling substrate access to the enzymes active site.

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Section: Resultsmentioning

confidence: 83%

Specific Inter-residue Interactions as Determinants of Human Monoacylglycerol Lipase Catalytic Competency

Tyukhtenko

Karageorgos

Rajarshi

et al. 2016

Journal of Biological Chemistry

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“…This identifies Cys (146) as a residue that may participate in the catalytic mechanism of the enzyme, as shown previously for the poliovirus 3C proteinase (Ivanoffet al, 1986). Apart from this Cys residue (in a -Gly-X-Cys-sequence) which is conserved together with a His residue (in position 160 for HRV-14) in the picornavirus proteinases, there is no further homology detectable between the 3C enzymes and papain or other members of the cysteine proteinase family (Berger & Schechter, 1970;Kamphuis et al, 1985 ;Polgar & Halasz, 1982). Therefore, it may well be that the picornavirus 3C proteinases represent a subclass of cysteine proteinases that are mechanistically similar to but homologous with papain.…”

Section: Discussionmentioning

confidence: 99%

Hydrolysis of a Series of Synthetic Peptide Substrates by the Human Rhinovirus 14 3C Proteinase, Cloned and Expressed in Escherichia coli

Orr¹,

Long²,

Kay³

et al. 1989

Journal of General Virology

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SUMMARYThe 3C proteins of several picornaviruses, including poliovirus, foot-and-mouth disease virus (FMDV) and encephatomyocarditis virus (EMCV), have been demonstrated to be cysteine-type proteinases, involved in the processing of the respective polyproteins expressed by the monocistronic RNA genome. Nucleotide sequencing data have indicated that the human rhinovirus 14 (HRV-14) RNA genome encodes a homologous 3C protein. The HRV-14 3C protein was purified to homogeneity from Escherichia coli expressing the cloned 3C genomic fragment. The enzyme was assayed against peptides corresponding to those residues, predicted (by nucleotide sequencing data) to occur at authentic cleavage sites within the polyprotein. The peptides representing the 1B/1C, 2A/2B, 2C/3A, 3A/3B, 3B/3C and 3C/3D cleavage sites, where proteolysis was predicted to occur at a Gln-Gly junction, were all cleaved by the 3C proteinase. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase. By contrast, peptides corresponding to the predicted 2B/2C and 1C/1D cleavage sites, where the processing was presumed to occur at a Gln-Ala or Glu-Gly bond respectively, were not cleaved by the 3C proteinase. The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys~Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV-14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile. The HRV-14 3C proteinase probably plays an important role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy.

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“…Among the amide bond-hydrolyzing enzymes, many utilize either the hydroxyl group of serine and threonine side chains or the thiol group of cysteine as a nucleophile to attack the scissile bond. Typical examples are the serine and cysteine proteases (25,26), the amydohydrolases, which catalyze the hydrolysis of asparagine and glutamine to their acidic forms (22,27,28), and the E. coli penicillin acylase responsible for the conversion of penicillin G to 6-aminopenicillanic acid (23). Interestingly, the N-carbamyl-D-amino-acid amidohydrolase from A. radiobacter shares about 25% identity with the aliphatic amidases of both Pseudomonas aeruginosa and Rhodococcus erythropolis (29,30) with the highest degree of homology being found in the region surrounding and including Cys 172 .…”

Section: Discussionmentioning

confidence: 99%

Topological Mapping of the Cysteine Residues of N-Carbamyl-D-amino-acid Amidohydrolase and Their Role in Enzymatic Activity

Grifantini

Pratesi

Galli

et al. 1996

Journal of Biological Chemistry

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The N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter NRRL B11291, the enzyme used for the industrial production of D-amino acids, was cloned, sequenced, and expressed in Escherichia coli. The protein, a dimer constituted by two identical subunits of 34,000 Da with five cysteines each, was susceptible to aggregation under oxidizing conditions and highly sensitive to hydrogen peroxide. To investigate the role of the cysteines in enzyme stability and activity, mutant proteins were constructed by site-directed mutagenesis in which the five residues were substituted by either Ala or Ser. Optically active D-amino acids have attained a wide variety of commercial applications as intermediates for the production of fine chemicals, including ␤-lactam antibiotics, peptide hormones, and pesticides (1, 2). In particular, D-phenylglycine and D-p-hydroxyphenylglycine are among the most important chiral building blocks for the production of semisynthetic penicillins and cephalosporins such as ampicillin and amoxicillin.Several optically active D-amino acids are currently produced in a two-step reaction process starting from D,L-5 monosubstituted hydantoins that are inexpensively synthesized from the corresponding aldehydes (3). In the first step, the substrate is hydrolyzed by a D-specific hydantoinase to give a D-carbamyl derivative. Subsequently, the carbamyl derivative is converted to the corresponding D-amino acid either by chemical methods (4) or by a second enzymatic step catalyzed by an N-carbamyl-D-amino-acid amidohydrolase (hereinafter carbamylase) (5). Because chemical methods have high reaction temperatures, low yields, long reaction times, and generate large amounts of waste, the enzymatic hydrolysis of the N-carbamyl derivatives is highly preferred. Indeed, the use of the D-hydantoinase plus carbamylase two-enzyme system is considered one of the most successful industrial applications of enzyme technology.Several microorganisms expressing both enzymatic activities have been isolated, and the optimal reaction conditions and the biochemical properties of the two enzymes have been studied in some detail (6, 7). From what has been published so far, it appears that both the activity and stability of the carbamylase are negatively affected by oxidizing conditions, suggesting that one or more cysteine residues are present in the enzyme. Indeed, the sensitivity of the enzyme to oxidizing conditions is one of the most serious drawbacks of the enzymatic D-amino acid production process, and a strict anaerobic regime is required to allow the completion of the substrate to product conversion (8).To shed light on the role of the cysteines in the activity and stability of this important industrial enzyme, we decided to study in detail the N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter NRRL B11291, a strain that is used industrially for D-amino acid production. In this paper we describe the characterization of the recombinant A. radiobacter N-carbamyl-D-amino-acid amidohydrolase expresse...

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Current problems in mechanistic studies of serine and cysteine proteinases

Cited by 164 publications

References 109 publications

Specific Inter-residue Interactions as Determinants of Human Monoacylglycerol Lipase Catalytic Competency

Specific Inter-residue Interactions as Determinants of Human Monoacylglycerol Lipase Catalytic Competency

Hydrolysis of a Series of Synthetic Peptide Substrates by the Human Rhinovirus 14 3C Proteinase, Cloned and Expressed in Escherichia coli

Topological Mapping of the Cysteine Residues of N-Carbamyl-D-amino-acid Amidohydrolase and Their Role in Enzymatic Activity

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