University of Groningen

Engineering specificity and activity of thermolysin-like proteases from Bacillusde Kreij, Arno

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M4 family specificity determinants

21

Substrate specificity in the highly heterogeneousM4 peptidase family is determined by a small

subset of amino acids.

AbstractThe members of the M4 peptidase

family are involved in processes as diverse aspathogenicity and industrial applications. Forthe first time a number of M4 family members,also known as thermolysin-like proteases(TLPs), has been characterized with an identicalsubstrate set and a uniform set of assayconditions. Characterization with peptidesubstrates, as well as HPLC analysis of β-caseindigests, shows that the M4 family is ahomogeneous family in terms of catalysis, eventhough there is a significant degree of aminoacid sequence variation. The results of thisstudy show that differences in substratespecificity within the M4 family do not correlatewith overall sequence differences but depend ona small number of identifiable amino acids.Indeed, molecular modeling, followed by sitedirected mutagenesis of one of the substratebinding pocket residues of the TLP of Bacillusstearothermophilus, converted the catalyticcharacteristics of this variant into that ofthermolysin.

IntroductionThermolysin-like proteases (TLPs) are

members of the peptidase family M4 (122) ofwhich thermolysin (TLN; EC 3.4.24.27) is theprototype. The phylogenetic tree for the M4family is shown in Fig. 2.1A. The familycontains only secreted eubacterialendopeptidases, from both Gram-positive andGram-negative sources. All members of thiscomprehensive family are produced as pre-pro-proteins. During export the pre-sequence(signal sequence) is cleaved off, whereas theprosequence has been shown to assist in proper

folding, by acting as a molecular chaperone(123). In addition, it has been shown that theprosequence can act as a specific inhibitor(124), thus preventing (125) unwantedproteolytic activity in the cytoplasm (123). Themature enzymes are all of moderate size, around35 kDa (316 amino acids for thermolysin).These proteases contain the typical HEXXHamino acid motif, require Zn2+ ions for theiractivity and contain multiple Ca2+ ions (up tofour) for stability. All enzymes are optimallyactive at neutral pH (122, 126).

For several of these enzymes three-dimensional structures are available (52, 54, 56,127). In fact, thermolysin was among the firstproteins for which the structure was solved.Although considerable sequence diversity existswithin this family (Fig. 2.1B), there is a highdegree of structural conservation. All membersfor which the structure has been solved, wereshown to consist of two major domains. The N-terminal domain contains mainly β-sheets,whereas the C-terminal domain predominantlycontains α-helices. The active site is located inthe cleft between these two domains. In thoseenzymes of which the structure has beendetermined, the catalytically essential Zn2+ ionis located at the bottom of this cleft (Fig. 2.2).In a significant number of published structuresin which TLN was co-crystallized withinhibitors (52, 57-61), the residues involved incatalysis could be identified.

The family also includes enzymes frompathogens such as Legionella, Listeria,Clostridium, Staphylococcus, Pseudomonas andVibrio. For example, pseudolysin, the TLPfrom Pseudomonas aeruginosa, has been shownto cause tissue damage by degrading collagens,

Chapter 2

22

1 M04.015 Bacillus thuringiensis TLP (-)2 M04.015 Bacillus cereus TLP (P05806)3 M04.015 Lactobacillus sp. TLP (Q48857)4 M04.015 Bacillus megaterium TLP (Q00891)5 M04.001 Bacillus caldolyticus thermolysin (P23384)6 M04.001 Bacillus sp. thermolysin (Q59223)7 M04.001 Alicyclobacillus acidocaldarius thermolysin (Q43880)8 M04.001 Bacillus stearothermophilus NprT protein (P06874)9 M04.001 Bacillus stearothermophilus NprS protein (P43133)10 M04.001 Bacillus thermoproteolyticus thermolysin (P00800)11 Brevibacillus brevis unassigned peptidase (M4) (P43263)12 Paenibacillus polymyxa unassigned peptidase (M4) (P29148)13 M04.012 Bacillus subtilis neutral protease B (P39899)14 Clostridium histolyticum unassigned peptidase (M4) (-)15 M04.011 Clostridium perfringens lambda toxin (Q46237)16 M04.009 Staphylococcus chromogenes aureolysin (AAF32312)17 M04.009 Staphylococcus epidermidis aureolysin (P43148)18 M04.014 Bacillus amyloliquefaciens bacillolysin (P06832)19 M04.014 Bacillus subtilis bacillolysin (P06142)20 M04.008 Listeria monocytogenes TLP (Listeria) (2) (P34025)21 M04.008 Listeria monocytogenes TLP (Listeria) (1) (P23224)

FigThseqphyfro/mC. thi

A

B

C

22 Streptomyces coelicolor unassigned peptidase (CAB66423)23 Erwinia carotovora unassigned peptidase (M4) (Q99132)24 M04.007 Enterococcus faecalis coccolysin (Q47786)25 Streptomyces coelicolor SC3D11.04C protein (CAB76001)26 Streptomyces griseus metallopeptidase II (-)27 Renibacterium salmoninarum unassigned peptidase (M4) (hemolysin) (P55111)28 M04.003 Vibrio anguillarum vibriolysin (P43147)29 M04.003 Vibrio proteolyticus vibriolysin (Q00971)30 M04.003 Vibrio vulnificus vibriolysin (O06694)31 M04.003 Vibrio cholerae vibriolysin (P24153)32 M04.016 Aeromonas hydrophila pro-aminopeptidase processing protease (-)33 M04.016 Aeromonas caviae pro-aminopeptidase processing protease (Q9R9S7)34 M04.005 Pseudomonas aeruginosa pseudolysin (P14756)35 M04.006 Legionella longbeachae vibriolysin hom. (P55110)36 M04.006 Legionella pneumophila vibriolysin hom. (P21347)37 Bacillus cereus unassigned peptidase (NprB protein) (CAB69809)38 Serratia marcescens unassigned peptidase (M4) (Q06517)

ure 2.1. A. Phylogenetic tree of peptidase family M4.e 5 peptidases used in this study are indicated. B. Key touences with Swiss Prot and MEROPS identifiers. Thelogenetic tree and key to the sequences were adapted

m the MEROPS database (http://www.merops.co.ukerops/famcards/m4.htm) with permission of the authors.Sequence identity matrix of the 5 peptidases used ins study.

M4 family specificity determinants

23

elastin and fibronectin (128), whereas the TLPsfrom Listeria spp. appear to be involved in thematuration of specific virulence factors (129).Furthermore, the active site organization of M4peptidases exhibits similarity to those of anumber of eukaryotic metallopeptidases, inparticular to members of the matrixmetalloproteases (MMP’s)(130). These latterenzymes were shown to be involved in anumber of important processes in man,including the processing of precursors that playmodulation roles in the formation of tumors. Inaddition, metallo-endopeptidases are involved inmany cellular processes such as exocytosis, cell-cell fusion and neuropeptide hydrolysis (131).Consequently, metalloproteases of the M4family have attracted increasing attention asmodel proteins for the development of specificinhibitors that can be applied for diseasetreatment (132). In addition, several members ofthis protease family are applied in industry, e.g.in baking, brewing and leather processing (24).Thermolysin is being used for the synthesis ofthe artificial sweetener aspartame (24).

In this study we have characterizedseveral thermolysin-like proteases (TLPs) ofBacillus and Staphylococcus species. Theavailability of an impressive amount ofsequence, structural and kinetic data renders thisgroup of proteases an ideal subject for rationaldesign strategies. Although some of the familymembers have been characterized individually(114, 115, 126, 133, 134) a consistentcomparison with an identical substrate set and auniform set of assay conditions has never beenconducted. Previously it was suggested thatTLPs exhibit a preference for large hydrophobicP1' residues (Leu or Phe) (114, 122, 130, 133).In addition, it has been demonstrated that the S1'pocket is the major determinant of the substratespecificity (114). Here we show that thethermolysin-like protease (TLP) family is anextremely homogeneous family in terms ofcatalysis, even though there is a significant

degree of sequence variation. Furthermore, weshow that existing differences in specificity andactivity between two individual members can becanceled by a single amino acid substitution.

Materials and MethodsGenetics.

The nprM gene encoding thermolysin(TLN) of B. thermoproteolyticus (135), the nprTgene encoding the TLP of B.stearothermophilus CU21 (136)(TLP-ste), thenprC gene encoding the TLP of B.cereus(124)(TLP-cer) and the nprB gene encoding theTLP of B.subtilis (137)(TLP-sub) were cloned,subcloned, and expressed as describedpreviously (80). The purified TLP ofStaphylococcus aureus (138)(Aureolysin orTLP-sau, EC 3.4.24.29) was kindly provided byDr. J. Potempa. Site-directed mutagenesis wasperformed by the PCR-based mega-primermethod, essentially as described by Sarkar andSommer (139). Mutagenic primers weredesigned such that mutant clones could berecognized by the appearance or disappearanceof an endonuclease restriction site (80). Thenucleotide sequences of mutated fragments ofthe nprT gene were verified by DNA sequenceanalysis.

Modeling and mutant design.A three-dimensional model of TLP-ste

was built on the basis of homology withthermolysin (86% sequence identity), using themolecular modeling program WHAT-IF (140).The modeling procedures have been describedin detail elsewhere (78). Because of the highsequence similarity the model was expected tobe sufficiently reliable for prediction andanalysis of the effects of most amino acidsubstitutions (78, 141). This has beenconfirmed by the fact that the model has beenused for the successful design of variousstabilizing mutations (43, 82, 142, 143).Throughout this paper, residues in all TLPs are

Chapter 2

24

numbered according to the numbering ofcorresponding residues in thermolysin.

Production and Characterization of Enzymes.Production and purification of the

enzymes were performed as described earlier(80, 144). Before determining the kineticparameters, protease preparations were desaltedto 20 mM NaAc pH 5.3, 5 mM CaCl2 and 20%isopropanol using pre-packed PD-10 gelfiltration columns supplied by AmershamPharmacia.

Specific activities of the TLPs towardscasein were determined according to a methodadapted from Fujii et al. (136): approximately0.5 µg of protease was incubated in 1 ml of 50mM 2-amino-2-(hydroxymethyl)-1,3-propane-diol (Tris⋅HCl) (pH 7.5) containing 0.8%(wt/vol) casein and 5 mM CaCl2 at 37 °C for 1h. The reaction was quenched by the additionof 1 ml of a solution containing 100 mM tri-chloro-acetic acid (TCA), pH 3.5. One unit ofactivity is defined as the amount of enzymeactivity needed to liberate a quantity of acid-soluble peptide corresponding to an increase inA275nm of 0.001 per min.

The kcat/Km and Km values forfurylacryloylated di- and tripeptides of theenzymes were determined at 37 °C in athermostated Perkin-Elmer Lambda 11spectrophotometer. The reaction mixture (1 ml)contained 50 mM Tris, 50 mM 4-morpholineethanesulfonic acid (MES) (pH 7.0),5 mM CaCl2, 5% DMSO, 0.5% 2-propanol,0.01% Triton X-100 and 100 µM to 2.5 mM ofsubstrate, and the reaction was followed bymeasuring the decrease in absorption at 345 nm(∆ε345 = -317 M-1⋅cm-1 )(114). All substrateswere supplied by Bachem. Stock solutions ofthe furylacryloylated dipeptides 3-(2-furylacryloyl)-L-glycyl-L-leucine-amide(FaGLa) and 3-(2-furylacryloyl)-L-glycyl-L-phenyl-amide (FaGFa), and of the

furylacryloylated tripeptides 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine(FaGLA) and 3-(2-furylacryloyl)-L-glycyl-L-phenylalanine-L-leucine (FaGFL) wereprepared by dissolving the peptides in Me2SO.The apparent second order rate constant kcat/Km

was determined by varying the enzymeconcentrations (over a 50-fold range) underpseudo-first-order conditions and measuring theinitial activity, essentially according to themethod described by Feder (114).

The Ki for N-[α-L-rhamnopyranosyl-oxyhydroxyphosphinyl]-L-leucine-L-tryptophan(phosphoramidon) was determined by a 10 minpreincubation of a 0.1 nM protease solutionwith varying concentrations of the inhibitor (10-

8 to 10-3 M), in 50 mM Tris, 50 mM MES (pH7.0), 5 mM CaCl2 , 0.01% Triton X100.Subsequently, enzyme activity was determinedusing 100 µM FaGLA as substrate. Ki ’s werecalculated by the method described by Hunterand Downs (145).

For the determination of thermalstability 0.1 µM purified protease solutions (in20 mM sodium acetate, pH 5.3, 5 mM CaCl2,0.01% Triton X-100, 0.5% 2-propanol, and 62.5mM NaCl) were incubated at varioustemperatures for 30 min, after which theresidual proteolytic activity was determinedwith casein as a substrate (136). Thermalstability was quantified by T50, being thetemperature giving 50% residual activity after a30 min period of incubation (78, 81).

The proteolytic properties of the mutantenzymes towards β-casein (Sigma-Aldrich),were determined by means of HPLC. β -casein(1 mg ml-1) was incubated in 50 mM Tris, 50mM MES (pH 7.0), 5 mM CaCl2, 0.01% TritonX-100 with each of the TLP variants at a molarratio of 1,000:1 at 37 °C for 24 hrs. Thepeptides resulting from hydrolysis werederivatized with dansyl-chloride. Theproteolytic products were separated by loading a

M4 family specificity determinants

25

sample corresponding to 50 µg β-casein on areversed phase column (RP-304, Bio-RadLaboratories). The mobile phase used was 50mM NaAc, pH 5.2. Peptides were eluted with alinear gradient of 0-60% acetonitrile in 30 minat a flow rate of 1 ml min-1. Absorption of theeluting peptides was monitored at 254 nm.

ResultsEnzymatic properties towards casein.

To investigate the activity of the variousM4 proteases on large protein substrates, theactivity towards casein was determined. Caseinwas selected as a standard substrate for activitymeasurements because it behaves as anoncompact and largely flexible structure (146),thus rendering all scissionable motifs accessibleto the same extent for the various proteases atall temperatures employed. Indeed, we haveshown previously that digestion of β-caseinwith TLP-ste at different temperatures yieldedidentical degradation products (84). The resultsare shown in Table II.I. Most of the wild-typeenzymes show similar specific activities, with avariation of a factor of approximately 3. Themajor exception is TLP-cer, which is much lessactive on casein than the other enzymes tested.

To determine the thermal stabilityand the optimal temperature for catalysisof the various proteases, we determinedthe T50 (79) values and the temperaturedependence of activity towards casein.The T50 values are given in Table II.I.These values correlate well with thetemperature optima of the TLPs as shownin Fig. 2.3, in the sense that the mostthermally labile protease shows the lowestoptimum temperature. To facilitatecomparison, the maximum activity of thedifferent TLPs has been normalized to100%.

Between closely related TLPs acorrelation exists between the degree of

sequence identity and the difference in thermalstability (see Table II.I for a comparison of thesequence identity and the ∆T50 values). In allcases, the temperature optimum is just belowthe T50 value determined, which is a direct resultof the experimental procedures: the T50 valueswere determined with a 30 min incubationperiod followed by determination of theremaining activity at 37 °C, whereas thetemperature optima were determined during a 1hr incubation period at the indicatedtemperatures. As a consequence, this longerincubation period can be expected to lead to ahigher degree of inactivation at the elevatedtemperatures.

Inspection of Fig. 2.3 shows that theshape of the curve of TLN differs as comparedto those of the other TLPs. Of the enzymestested only TLP-sub and TLN shows Arrheniusbehavior: the activity increases exponentiallywith the temperature. TLP-sau deviates fromthe other TLP’s by showing an unexpectedlybroad temperature optimum, suggesting thatthermal (in)activation of this protease mightdiffer from that in the other proteases.

Table II.I Specific activity and thermostability of TLP’s.

SpecificActivitya

T50b ∆T50

to TLNSequence identityto TLN

Units × 10-3 °C °C °C

TLP-cer 1.4 68.6 18.3 73TLP-ste 29 73.4 13.5 86TLN 42 86.9 0.0 100TLP-sub 47 58.6 28.3 48TLP-sau 88 55.1 31.8 45

TLP-ste F133L

57 74.9 12.0 86

aStandard deviations are less than 10 % of the values given.bThe error margin amounts to approximately 0.3 ºC.

Chapter 2

2

Ct

spwaItPTrsT

shown by the Phe/Leu ratio for dipeptides. Incontrast, the M4 family is often described ashaving an equal P1' preference for Leu and Phe(114, 122, 130, 133). The diversity or similarityin primary amino acid sequence (Fig 2.1B), is

Fsthαt(

igure 2.2 Ribbons diagram of thermolysin. The leftide shows the predominantly β-sheet containing N-erminal domain; the right side the predominantly α-elical C-terminal domain. The centre shows the central-helix which is the bottom of the active site cleft with

he catalytic Zn2+ ion (large sphere). Four Ca2+ ionssmall spheres), involved in stability, are also shown.

6

atalytic properties of TLPs on di- andripeptide substrates.

To determine the P1' substratepecificity, the activities of the various M4roteases towards di- and tripeptide substratesere determined. Their activities on dipeptide

nd tripeptide substrates are shown in TablesI.II and II.III, respectively. The results showhat both substrates with a Leu as well as with ahe as P1' residue are efficiently hydrolyzed byLPs. As with casein, TLP-cer shows a

elatively low activity towards dipeptideubstrates. With dipeptide substrates, mostLPs prefer Leu over Phe at the P1' position, as

Figure 2.3 Temperature optima of TLPs determinedwith casein as substrate. The maximum activity of eachTLP is set as 100%. ΟTLP-cer, �TLP-ste, TLN,�TLP-sub, ∆TLP-sau, �TLP-ste F133L

M4 family specificity determinants

27

not reflected in either different or similarcleavage efficiencies for the peptide substratestested. In fact, the substrate pecificity of TLN ismuch more similar to that of TLP-sub than tothat of TLP-ste, contrary to what might beexpected on the basis of sequence similarity

(45% and 86% identity, respectively). With theexception of TLP-cer, all activities on peptidesubstrates are less than one order of magnitudedifferent from those of TLN.

In contrast, the inhibition constant forthe inhibitor phosphoramidon, which wasspecifically designed for TLN, seems tocorrelate with the sequence difference. TheTLPs that are phylogenetically close to TLN aremuch more sensitive to phosphoramidon ascompared to those that are more distant.

HPLC characterization of β-casein digests.To examine whether differences in

substrate specificity can be observed on largepeptide substrates, HPLC analyses wereperformed on β-casein hydrolysates by thevarious TLPs. Fig. 2.4 shows a detail of the RP-HPLC analyses of the peptides that were formedupon digestion of β-casein with the TLPs. Anumber of characteristic and reproducibleproducts could be identified for each of theTLPs. Although the preference towards thesmall peptides used showed little variation,differences in the digestion patterns of β-caseinare clearly detectable. This illustrates thatdifferences can be much more readily detectableon large protein substrates than on small peptidesubstrates.

Table II.III Activity of TLPs on tripeptide substrates.

kcat/KM PheFaGLAa FaGFLa Leu

s-1⋅M-1 × 10-5 ratio kcat/KM

TLP-cer 0.17 0.56 3.4TLP-ste 0.56 8.30 14.9TLN 2.05 3.42 1.7TLP-sub 0.79 0.42 0.5TLP-sau 0.18 0.33 1.8

TLP-ste-F133L 1.77 4.34 2.5

aStandard deviations are less than 15 % of the valuesgiven.

Table II.II Activity of TLPs on dipeptide substratesand inhibition constants for phosphoramidon.

kcat/KM Phe Kib

FaGLaa FaGFaa Leu Inhibitionc

s-1⋅M-1 × 10-3 ratio kcat/KM nM

TLP-cer 0.64 0.46 0.7 2.1x102

TLP-ste 2.20 3.40 1.5 13TLN 12.25 3.88 0.3 21TLP-sub 3.20 0.17 0.1 6.1x103

TLP-sau 3.47 5.48 1.6 3.0x103

TLP-ste F133L 17.15 6.31 0.4 17

aStandard deviations are less than 15 % of the valuesgiven. b Phosphoramidon. cStandard deviations are lessthan 10 % of the values given.

Figure 2.4. Detail of HPLC diagrams of ββββ-caseindigests. 1. TLP-ste, 2. TLP-ste F133L, 3. TLN, 4. TLP-sub, 5. TLP-sau, 6. TLP-cer

Chapter 2

28

Site

diffvaridissdiffthe modiffmemandare conlatteextrfeatThearouthatcommofeatdiff

FLs

igure 2.5. Stereo view line drawing of the S1' pocket, showing the P1' Leu side chain, coordinated by theeu202 and the 133 residue. Residue 133 is a Leu in TLN and a Phe in TLP-ste. Mutation Phe133Leu in TLP-

te is predicted to change the S ’ specificity of TLP-ste into that of TLN.

-directed mutagenesis of the active site.The results presented above suggest that

erences in substrate specificity between TLPants are not correlated with overall sequenceimilarities. To examine whether sucherences might be reflected in the structure ofactive site and substrate binding pockets,

lecular modeling of the active sites of theerent variants was employed. For several

bers of the family (TLN, TLP-cer, TLP-sau elastase) high resolution X-ray structuresavailable. In addition, models have been

structed for TLP-sub and TLP-ste. Ther models have previously been shownemely useful for identifying structuralures involved in thermal stability (78, 84). fact that the amino acid conservation in andnd the active sites is very high, suggests

they are structurally similar. We decided topare the active sites of TLN and TLP-ste in

re detail in order to identify structuralures that could explain the observederences in substrate specificity. The two

enzymes are highly similar (86 % sequenceidentity). In particular, in the active site regionsthe sequence conservation is very high.Therefore, the constructed model for TLP-ste isexpected to be highly reliable in this region.

Close inspection of the model of TLP-ste and careful comparison with the TLNstructures available, revealed that one of themajor differences between the two TLPs in theactive site region concerns residue 133, which isa Leu in TLN but a Phe in TLP-ste. The S1'subsite is composed of the side chains ofPhe130, Phe/Leu133, Val139 and Leu202.Furthermore, inspection of the S1' pocket andthe conformation of the various P1' side chainsin TLN-inhibitor complexes (1TLP.PDB, 1-7TMN.PDB, 4-8TLN.PDB) shows that the P1'side chain is sandwiched between the 133 andthe 202 S1' residues (Fig. 2.5).

It might be anticipated that the largePhe133 residue in the S1' pocket will influencethe binding of substrates in this specificity-determining pocket to a considerable extent. Totest this hypothesis, Phe133 in TLP-ste was

1

M4 family specificity determinants

29

substituted by Leu and the effects on substratespecificity were determined. As documented inTables II.II and II.III, the TLP-ste mutant showsenzymatic characteristics on di- and tripeptidesthat are much more TLN-like than TLP-ste-like.In addition, this mutation almost doubled theactivity towards casein (Table II.I).Furthermore, the RP-HPLC patterns obtainedwith the TLP-ste F133L mutant showed someTLN-specific peaks, whereas some TLP-stespecific peak continue to be present as well.However, the temperature optimum, the shapeof the temperature curve and the thermalstability of the single mutant remained identicalto wild-type TLP-ste.

DiscussionThe present study shows a correlation

between the thermal stability and sequenceidentity of the various TLPs. This correlationwith sequence identity does not exist for thedifferences in activity and specificity on bothpeptide substrates and casein. Althoughdifferences in specificity on the peptidesubstrates used are relatively small, HPLCanalysis of digestion patterns of β-casein doesshow specific digestion patterns. The fact thatthe differences in activity and specificity can becanceled by mutating one amino acid in asubstrate binding pocket, indicates that notoverall sequence differences but a small set ofidentifiable amino acid residues is responsiblefor the differences in performance of theseenzymes.

The comparison of the thermostability ofclosely related TLPs, showed that a correlationexists between the sequence identity and thedifference in thermal stability. However,inspection of Fig. 3 shows that TLP-sub andTLN are the only two enzymes of which thethermal activation shows Arrhenius likebehavior. A previously described hyperstablevariant of TLP-ste (84) does not show thisbehavior (147). Thus it seems unlikely that

thermal stability underlies this differencebetween these two and the remainder of theenzymes studied. Rather a process such ashinge-bending (89, 148) could be a more likelycause for the difference between these two setsof enzymes.

The comparison of the enzymaticperformance, i.e. activity and specificity, ofTLPs from Bacilli and Staphylococcus indicatesthat overall divergence in primary sequence isnot correlated with differences in activity andsubstrate specificity. In contrast, local sequencedifferences in the active site and bindingpockets seem to be responsible for the majorityof the differences in activity and substratespecificity. This hypothesis is supported by theobservation that both the kcat/Km for the di- andtripeptide substrates differ less than 1 order ofmagnitude between the various enzymes, withthe exception of TLP-cer, and, as shown by theTLP-ste F133L mutant, the observation that theactivity and substrate specificity of one variantcan be changed into that of another by mutatingjust one binding pocket residue. However, thishypothesis seems to be contradicted by theapparent relation between the Ki forphosphoramidon and the sequence difference.This relation can be explained by the fact thatthe most important residue for phosphoramidonbinding is Phe114 (52, 61, 149) present in bothTLN and TLP-ste (low Ki) whereas the 114position in TLP-cer, -sub and –sau is occupiedby an Ala (high Ki).

The similarity in activity and specificityof the various TLP’s towards peptide substratesdoes not exclude the possibility that overallsequence differences can play a role in activityand specificity towards larger proteinaceoussubstrates. Analysis of the digestion patterns ofβ-casein indicated that there are cleardifferences in substrate specificity on proteinsubstrates. However, the mutant TLP-steF133L, which changes the specificity of TLP-ste to that of TLN on peptide substrates, also

Chapter 2

30

changes the digestion pattern on β-casein into amore TLN like pattern. Although otherexplanations cannot be excluded, this suggeststhat the observed differences in specificity aremainly caused by differences in the active siteand binding pockets and not by overall sequencedifferences.

The present study is the first example ofan approach in which the enzymatic andcatalytic properties of a significant number ofmembers of the M4 peptidase family arecompared under identical conditions. The needof such a comparison is obvious, in view of theroles of members of this family in processes asdiverse as pathogenicity and industrialapplications. The notion that overall differencesin sequence do not correlate with substratespecificity enabled us to modify the substrate

specificity by site-directed mutagenesis of thoseresidues directly involved in substrate bindingand catalysis. Indeed, a single amino acidsubstitution converted catalytic characteristicsof one family member into that of another.Consequently it can be envisaged that specificinhibitors, for example to be used for blockingdisease-related members of the MMP family,can be designed on the basis of amino acidresidues identified in TLPs. Thus, this studyprovides additional arguments for the potentialof TLPs as a model system in the search fornovel MMP inhibitors. Both for thedevelopment of specific inhibitors as well as forthe improvement of biocatalysts, a betterunderstanding of existing relations betweensequence, structure and function is ofconsiderable importance.