Novel kinetic analysis of enzymatic dipeptide synthesis: Effect of pH and substrates on thermolysin catalysis

Murakami, Yoshihiko; Chiba, Kazuyuki; Oda, T.; Hirata, Akira

doi:10.1002/bit.1131

Cited by 3 publications

(3 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, in absence of Mpl, processing of PC-PLC S51D S53N was undetectable. These results suggest that Mpl is responsible for the proteolytic activation of PC-PLC S51D S53N, even though D and N are not generally favorable for protease activity of the Mpl family (23).…”

Section: Discussionmentioning

confidence: 84%

The Metalloprotease ofListeria monocytogenesControls Cell Wall Translocation of the Broad-Range Phospholipase C

2005

View full text Add to dashboard Cite

Listeria monocytogenes is a gram-positive bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from secondary vacuoles. PC-PLC requires cleavage of an N-terminal propeptide for activation, and Mpl, a metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. Previously, we showed that cell wall translocation of PC-PLC is inefficient, resulting in accumulation of PC-PLC at the membrane-cell wall interface. In infected cells, rapid cell wall translocation of PC-PLC is triggered by a decrease in pH and correlates with cleavage of the propeptide in an Mpl-dependent manner. To address the role of the propeptide and of Mpl in cell wall translocation of PC-PLC, we generated a cleavage site mutant and a propeptide deletion mutant. The intracellular behavior of these mutants was assessed in pulse-chase experiments. We observed efficient translocation of the proform of the PC-PLC cleavage site mutant in a manner that was pH sensitive and Mpl dependent. However, the propeptide deletion mutant was efficiently translocated into host cells independent of Mpl and pH. Overall, these results suggest that Mpl regulates PC-PLC translocation across the bacterial cell wall in a manner that is dependent on the presence of the propeptide but independent of propeptide cleavage. In addition, similarly to Mpl-mediated cleavage of PC-PLC propeptide, Mpl-mediated translocation of PC-PLC across the bacterial cell wall is pH sensitive.

show abstract

Section: Discussionmentioning

confidence: 84%

The Metalloprotease ofListeria monocytogenesControls Cell Wall Translocation of the Broad-Range Phospholipase C

2005

View full text Add to dashboard Cite

show abstract

“…Neprilysin inhibitors have attracted great interest as they show antinociceptive and antihypertensive properties . Due to its high thermostability, tolerance to organic solvents, and specifity against hydrophobic residues, TLN is also used in industrial processes, for example, as a catalyst for the hydrolysis and synthesis of a precursor of aspartame and in the continuous synthesis of peptide derivates (see refs in ref ).…”

Section: Introductionmentioning

confidence: 99%

Peptide Hydrolysis in Thermolysin: Ab Initio QM/MM Investigation of the Glu143-Assisted Water Addition Mechanism

Blumberger

Lamoureux

Klein

2007

J. Chem. Theory Comput.

View full text Add to dashboard Cite

Thermolysin (TLN) is one of the best-studied zinc metalloproteases. Yet the mechanism of action is still under debate. In order to investigate the energetic feasibility of the currently most favored mechanism, we have docked a tripeptide to the active site of TLN and computed the free energy profile at the quantum mechanics/molecular mechanics level of theory. The mechanism consists of three distinct steps: (i) a Zn-bound water molecule is deprotonated by Glu143 and attacks the carbonyl bond of the substrate; (ii) Glu143 transfers the proton to the amide nitrogen atom; (iii) the nitrogen atom is protonated and the peptide bond is irreversibly broken. The free energy barriers for steps i and iii have almost equal heights, 14.8 and 14.7 kcal/mol, respectively, and are in good agreement with the effective experimental activation barrier obtained for similar substrates, 12.1-13.6 kcal/mol. Transition state stabilization for nucleophilic attack is achieved by formation of a weak coordination bond between the substrate carbonyl oxygen atom and the Zn ion and of three strong hydrogen bonds between the substrate and protonated His231 and two solvent molecules. The transition state for the nucleophilic attack (step i) is more tightly bonded than the enzyme-substrate complex, implying that TLN complies with Pauling's hypothesis regarding transition-state stabilization. Glu143, at first unfavorably oriented for protonation of the amide nitrogen atom, displayed large structural fluctuations that facilitated reorganization of the local hydrogen-bond network and transport of the proton to the leaving group on the nanosecond time scale. The present simulations give further evidence that Glu143 is a highly effective proton shuttle which should be assigned a key role in any reaction mechanism proposed for TLN.

show abstract

“…The substrate specificities of BLN, WT and TLN in dipeptide synthesis were compared with respect to amino acid residues in P ′ 1 position. In hydrolysis, TLN and the neutral protease from B. stearothermophilus are known to prefer bulky hydrophobic amino acids [23,24]. The same reaction conditions as for the synthesis of Z‐Asp‐Phe‐OMe were used, but the amino component Phe‐OMe was replaced with Ala‐OMe, Ile‐OMe, Leu‐OMe, Met‐OMe, Tyr‐OMe, Phe‐OMe or Val‐OMe; Table 1 summarizes the initial rates for the corresponding reactions.…”

Section: Resultsmentioning

confidence: 99%

Boilysin and thermolysin in dipeptide synthesis: a comparative study

Kühn

Dürrschmidt

Mansfeld

et al. 2002

Biotech and App Biochem

View full text Add to dashboard Cite

Boilysin (BLN) is an engineered, highly thermostable neutral protease from Bacillus stearothermophilus. Its high resistance is based on the stabilization of a surface loop (amino acid residues 55-69) by eight amino acid exchanges, including the introduction of a disulphide bond. In the present study, BLN was compared with the well-known and structurally related thermolysin (from B. thermoproteolyticus) with respect to their dipeptide- synthetic properties. The synthesis of the aspartame precursor N-(benzyloxycarbonyl)-l-aspartyl-l-phenylalanine methyl ester (Z-Asp-Phe-OMe) was used as the model reaction to study its enantioselectivity as well as the influence of neutral salt, temperature and calcium- ion concentration on peptide synthesis. The reactions were performed in homogeneous reaction systems containing DMSO or aliphatic alcohols. Furthermore, the substrate specificity in the synthesis of Z-Asp-X-OMe (X=Phe, d-Phe, Ala, Ile, Leu, Met, Tyr or Val) was examined. The two enzymes showed no difference in time course of reaction by the use of salts as activators or different alcohols as co-solvents. Both enzymes showed a high enantioselectivity towards the amino component in the reaction. They were strongly activated by NaCl, whereas the final product yields were strongly decreased at high NaCl concentrations. Aliphatic alcohols act as an inhibitor, but allow higher product yields compared with purely aqueous medium. Differences between the two enzymes are found at higher temperatures (>/=60 degrees C) in the absence or at low concentrations of Ca(2+) ions; BLN proved superior under these conditions, because its stability is less dependent on Ca(2+) ions. The substrate specificities of BLN and thermolysin at the P(')(1) position follow the same tendencies, with differences in the initial rates for the conversion of Z-Asp with Ile-OMe, Leu-OMe and Val-OMe.

show abstract

Novel kinetic analysis of enzymatic dipeptide synthesis: Effect of pH and substrates on thermolysin catalysis

Cited by 3 publications

References 41 publications

The Metalloprotease ofListeria monocytogenesControls Cell Wall Translocation of the Broad-Range Phospholipase C

The Metalloprotease ofListeria monocytogenesControls Cell Wall Translocation of the Broad-Range Phospholipase C

Peptide Hydrolysis in Thermolysin: Ab Initio QM/MM Investigation of the Glu143-Assisted Water Addition Mechanism

Boilysin and thermolysin in dipeptide synthesis: a comparative study

Contact Info

Product

Resources

About