Propeptide of the metalloprotease of Brevibacillus brevis 7882 is a strong inhibitor of the mature enzyme

Abstract

A metalloprotease gene of Brevibacillus brevis (npr) was expressed in Escherichia coli in a soluble form as native Npr precursor. A significant fraction of the precursor was spontaneously processed, producing the N-terminal propeptide and the mature enzyme. A strong inhibition of the mature Npr by its own propeptide in the crude lysate was observed even in the absence of the covalent linkage between them. Pure precursor, propeptide and the mature Npr were isolated and kinetic parameters of the mature enzyme inhibition by the propeptide were determined. The inhibition is of the tight-binding competitive type with K i 0.17 nM. Inhibition of metalloproteases from Brevibacillus megaterium and thermolysine by the heterologous propeptide of the Npr from B. brevis was much weaker or none.z 1999 Federation of European Biochemical Societies.Key words: Proenzyme; Tight-binding inhibition ; Brevibacillus brevis IntroductionMany proteases in various organisms are synthesized as inactive precursors, preproproteins or zymogens. Generally it is almost impossible to isolate a bacillar protease precursor from a natural strain, since it appears in the cultural medium as a processed mature form. When expressed in E. coli or other Gram-negative bacteria, these proteins are accumulated in insoluble inclusion bodies formed by non-active precursor, as found for subtilisin E [1], K-lytic protease from Lysobacter enzymogenes [2] and yeast carboxypeptidases Y [3], or they get rapidly processed producing mature form, as in the case of elastase from P. aeruginosa [4] and K-lytic protease from L. enzymogenes [2]. Therefore protease expression in a form of the non-active precursor is a challenging task. For that reason protease precursors and mechanisms of there activation were not thoroughly studied.The presequences usually serve as signal peptides for a transport through the plasma membrane. A functional role of the propeptides is less clear. Several functions were proposed for the propeptides. The propeptide may be required for productive folding and/or maintaining the protease in an inactive state inside the cell, as well as for interaction with the transport machinery of the cell which is important for e¡ec-tive secretion of these proteins [5].The role of protease prosequences is most extensively studied in the serine proteases: subtilisin E from B. subtilis [6,7], K-lytic protease from L. enzymogenes [8] and the vacuolar carboxypeptidase Y from yeast S. cerevisiae [3]. In all mentioned examples the propeptide assists the folding of the respective enzymes in vivo and in vitro, and blocks the enzymatic activity when covalently attached to the`mature' moiety of the enzyme. In subtilisin E and K-lytic protease their propeptides appear as tight-binding inhibitors of mature enzymes in trans. A propeptide of carboxypeptidase Y has a low a¤n-ity for its mature enzyme in vitro. The propeptides of cystein proteases papain and cathepsin B are found to inhibit their respective mature enzymes [9,10]. In a cystein vacuolar protease cathepsi...

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

“…The results indicate that the propeptide inhibits the enzyme competitively with a K i value of 0.027 M. The propeptide had no inhibitory effect on porcine pepsin when examined under similar conditions (data not shown). Table I 18 -Tyr 25 were incubated with the enzyme for a longer period. Under the same conditions, however, the 8-residue peptide Lys-Arg-His-SerAsn-Ala-Ala-Tyr, which had been derived from the central 8-residue peptide Lys 18 -Tyr 25 (peptide 22) by replacing the Pro-Pro sequence with an Ala-Ala sequence, was found to be hydrolyzed by the enzyme at pH 4.0 at the His-Ser bond (extent of hydrolysis: ϳ20% in 3 h under the conditions used) (data not shown).…”

“…Lys 18 and Arg 14 contributed to lesser extents to the inhibition. These results will be useful for developing effective peptide-based inhibitors for the enzyme.…”

“…These T m values, however, did not change when they were incubated with the propeptide under the same conditions (data not shown). 18 -Tyr 25 -The denaturation curves of the enzyme in the presence and absence of some typical truncated propeptides and Ala scan-substituted peptides of Lys 18 -Tyr 25 are shown in Fig. 5.…”

“…It may be also possible that the latter pK a values reflect the deprotonation of the side chain of His 20 . It is interesting that the double substitution, but not the single substitution, of the two prolines in Lys 18 -Tyr 25 with alanine changed the inhibitor to a substrate. Presumably, the mode of binding of the peptide would be changed to a productive one through a subtle change in the peptide/protein interaction induced by these substitutions, whereas the inhibitor with the two prolines should bind to the enzyme in a nonproductive mode.…”

“…These roles, however, have not yet been fully understood, and further studies on various proteins are necessary. As for the inhibitory properties of the propeptides of peptidases, there are numbers of reports describing their inhibition profiles and type of inhibition (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). However, only a few attempts have been made so far to identify critical sequences and/or residues in the propeptide important for inhibition of the corresponding mature peptidase, and much of the inhibition mechanisms, the structural determinants in the propeptide, and the sites of binding involved in the inhibition remains to be established.…”