The Role of Histidine 231 in Thermolysin-like Enzymes.

Beaumont, Ann; O’Donohue, Michael; Paredes, Nathalie; Rousselet, Nathalie; Assicot, M.; Bohuon, C; Fournié‐Zaluski, Marie‐Claude; Roques, Bernárd P.

doi:10.1074/jbc.270.28.16803

Cited by 74 publications

(61 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Residue in Human ACE C-domain-In thermolysin, thermolysinlike enzymes, and neprilysin, in which the identity of the catalytic His has been confirmed by site-directed mutagenesis (12,13) and x-ray crystallography (14,15), this residue is found C-terminal to the EXXD zinc-binding motif and is preceded by a conserved Asp with 1 or 4 intervening residues. The distance between the EXXD motif and the transition state His is 49 -60 residues in vertebrate neprilysin-like and bacterial thermolysin-like sequences.…”

Section: Prediction Of His 1089 As a Transition State Stabilizingmentioning

confidence: 99%

“…For example, retro-inverso modification of the amide bond (from -CONH-to -NHCO-) has little effect on inhibitory potencies of mercaptan inhibitors of thermolysin and neprilysin, but this change produces a marked decrease (Ͼ1,000-fold) in ACE inhibitor potency (12,29). These findings illustrate differences in the substrate binding pocket of these gluzincins distal to the site that interacts with the scissile carbonyl bond.…”

Section: Prediction Of His 1089 As a Transition State Stabilizingmentioning

confidence: 99%

See 1 more Smart Citation

Angiotensin I-converting Enzyme Transition State Stabilization by His1089

Fernandez

Liu

Wouters

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N-and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His 1089 as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His 1089 to Ala or Leu. At pH 7.5, with Ang I as substrate, k cat /K m values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wildtype enzyme and were mainly due to a decrease in catalytic rate (k cat ) with minor effects on ground state substrate binding (K m ). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His 1089 3 Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His 1089 side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His 1089 on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.

show abstract

Section: Prediction Of His 1089 As a Transition State Stabilizingmentioning

confidence: 99%

Section: Prediction Of His 1089 As a Transition State Stabilizingmentioning

confidence: 99%

Angiotensin I-converting Enzyme Transition State Stabilization by His1089

Fernandez

Liu

Wouters

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The members of the thermolysin family are synthesized as inactive precursors, with a large N-terminal propeptide acting as a specific inhibitor for the mature protease and/or an intramolecular chaperone to control the folding of the protease (10,14,20,24). Since the N-terminal propeptide is cleaved by autoproteolysis (9,13,24), a transformant carrying a cloned metalloprotease gene also produces a correctly processed metalloprotease (2,13). On the other hand, studies of metalloproteases from vibrios (4,6,15) have demonstrated that, although these also belong to the thermolysin family, their precursors have C-terminal propeptides as well as N-terminal ones.…”

mentioning

confidence: 99%

Functional domains of a zinc metalloprotease from Vibrio vulnificus

et al. 1997

View full text Add to dashboard Cite

Vibrio vulnificus, an opportunistic human pathogen causing wound infection and septicemia, secretes a 45-kDa metalloprotease (V. vulnificus protease; VVP). A plasmid which carries the entire vvp gene subcloned into pBluescriptIIKS؉ was transformed into Escherichia coli DH5␣ for overproduction of the protease. The 45-kDa recombinant protease (rVVP) was isolated from the periplasmic fraction of the transformant by ammonium sulfate precipitation followed by column chromatography on phenyl Sepharose. Biochemical characterization of the isolated rVVP showed that the recombinant protease was identical to that produced by V. vulnificus. When rVVP was incubated at 37°C, a 35-kDa fragment was generated through autoproteolytic removal of the C-terminal peptide. This 35-kDa fragment (rVVP-N) was found to have sufficient proteolytic activity toward oligopeptides and soluble proteins but had markedly reduced activity toward insoluble proteins. LineweaverBurk plot analysis indicated increased K m values of rVVP-N for all of the protein substrates. rVVP, but not rVVP-N, was shown to agglutinate rabbit erythrocytes, bind to the erythrocyte ghosts, and digest the ghost membrane proteins. These results strongly suggest that rVVP (and VVP) consists of at least two functional domains: an N-terminal 35-kDa polypeptide mediating proteolysis and a C-terminal 10-kDa polypeptide which may be essential for efficient attachment to protein substrates and erythrocyte membranes.Metalloproteases, in which zinc is an essential metal ion for catalytic activity, are elaborated by various human pathogenic bacteria, as well as by nonpathogenic ones (5). They can be classified into three families: thermolysins, serralysins, and neurotoxins (7). The members of the thermolysin family are synthesized as inactive precursors, with a large N-terminal propeptide acting as a specific inhibitor for the mature protease and/or an intramolecular chaperone to control the folding of the protease (10,14,20,24). Since the N-terminal propeptide is cleaved by autoproteolysis (9, 13, 24), a transformant carrying a cloned metalloprotease gene also produces a correctly processed metalloprotease (2, 13). On the other hand, studies of metalloproteases from vibrios (4,6,15) have demonstrated that, although these also belong to the thermolysin family, their precursors have C-terminal propeptides as well as N-terminal ones. The biochemical function of the Cterminal propeptide is currently unknown.Vibrio vulnificus is an opportunistic human pathogen causing wound infection and septicemia (8,22). Like other vibrios, this pathogen secretes the 45-kDa zinc metalloprotease (V. vulnificus protease; VVP) since it belongs to the thermolysin family (18). VVP has been reported to have many biological functions: proteolytic degradation of a wide variety of host proteins, such as plasma proteins, involved in coagulation or complement action, induction of hemorrhagic tissue damage, and enhancement of vascular permeability through the generation of inflammatory mediators (18). Recently, ...

show abstract

“…The new catalytic mutant was generated by mutating H437, an active site histidine involved in stabilizing the transition state during catalysis (3). We also inverted the tags to eliminate the possibility that either tag interfered with substrate binding or catalysis.…”

Section: Resultsmentioning

confidence: 99%

“…The active site zinc ion of these enzymes is coordinated by a water molecule and three amino acid residues, including the two histidines present within the HEXXH motif and a glutamic acid located 20 residues downstream of this motif (2,8). In addition, the glutamic acid residue located within the HEXXH motif and a histidine residue located 83 residues downstream of this motif interact with a water molecule at the active site and are required for catalysis (2,3).…”

mentioning

confidence: 99%

The Metalloprotease ofListeria monocytogenesIs Activated by Intramolecular Autocatalysis

2008

View full text Add to dashboard Cite

The metalloprotease (Mpl) of Listeria monocytogenes is a thermolysin-like protease that mediates the maturation of a broad-range phospholipase C, whose function contributes to the ability of this food-borne bacterial pathogen to survive intracellularly. Mpl is made as a proprotein that undergoes maturation by proteolytic cleavage of a large N-terminal prodomain. In this study, we identified the N terminus of mature Mpl and generated Mpl catalytic mutants to investigate the mechanism of Mpl maturation. We observed that Mpl activity was a prerequisite for maturation, suggesting a mechanism of autocatalysis. Furthermore, using a strain of L. monocytogenes expressing both the wild-type form and a catalytic mutant form of Mpl simultaneously, we determined that in vivo maturation of Mpl occurs exclusively by an intramolecular autocatalysis mechanism.Listeria monocytogenes is a facultative intracellular bacterial pathogen that multiplies in the cytosol of host cells and uses an actin-based mechanism of motility to spread from cell to cell without exiting the intracellular milieu (26). Upon cell-to-cell spread, bacteria are temporarily located in double-membrane vacuoles from which they must exit to perpetuate the intracellular life cycle. Among the factors contributing to vacuolar lysis is a bacterial phospholipase C (PC-PLC) (24, 28). PC-PLC is made as a proenzyme whose activation requires cleavage of a 24-amino-acid propeptide. The metalloprotease of Listeria (Mpl) contributes to PC-PLC activation (21). Moreover, Mplmediated maturation of PC-PLC is regulated in a temporal and spatial manner during the intracellular life cycle of L. monocytogenes (15).Mpl possesses the HEXXH motif that is characteristic of the Zincins superfamily of metalloproteases. Within this family, Mpl is most closely related to thermolysin, which is the prototype member of the M4 family of metalloproteases (20). Thermolysin is made by Bacillus thermoproteolyticus, and all members of the thermolysin family originate from bacteria. The active site zinc ion of these enzymes is coordinated by a water molecule and three amino acid residues, including the two histidines present within the HEXXH motif and a glutamic acid located 20 residues downstream of this motif (2, 8). In addition, the glutamic acid residue located within the HEXXH motif and a histidine residue located 83 residues downstream of this motif interact with a water molecule at the active site and are required for catalysis (2, 3).Thermolysin and related metalloproteases are synthesized as preproenzymes. The prodomain, which accounts for ϳ40% of the proenzyme, serves as a chaperone and as an inhibitor of catalysis (17,23,29). Processing of the prodomain generates the mature active form of the protease. Autocatalysis has been suggested to be the mechanism of maturation of thermolysinlike proteases, as catalytic site mutants fail to generate mature proteases (10, 11, 13, 16). Furthermore, a mechanism of intramolecular autocatalysis was suggested for thermolysin itself, as purified active t...

show abstract

The Role of Histidine 231 in Thermolysin-like Enzymes.

Cited by 74 publications

References 42 publications

Angiotensin I-converting Enzyme Transition State Stabilization by His1089

Angiotensin I-converting Enzyme Transition State Stabilization by His1089

Functional domains of a zinc metalloprotease from Vibrio vulnificus

The Metalloprotease ofListeria monocytogenesIs Activated by Intramolecular Autocatalysis

Contact Info

Product

Resources

About