Ficin: a cysteine proteinase with binding site-catalytic site signalling characteristics intermediate between those of papain and actinidin

1993 ◽  
Vol 21 (2) ◽  
pp. 216S-216S
Author(s):  
Manij PATEL ◽  
Mark P. THOMAS ◽  
Irfan S. KAYANI ◽  
Geoffrey W. MELLOR ◽  
Emrys W. THOMAS ◽  
...  
Keyword(s):  
Binding Site ◽  
Cysteine Proteinase ◽  
Catalytic Site

scholarly journals A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation

2020 ◽  
Vol 295 (28) ◽  
pp. 9474-9489 ◽  
Author(s):  
Manon Molina ◽  
Claire Moulis ◽  
Nelly Monties ◽  
David Guieysse ◽  
Sandrine Morel ◽  
...  
Keyword(s):  
Binding Site ◽  
Molar Mass ◽  
Enzyme Stability ◽  
Catalytic Site ◽  
Glycoside Hydrolase Family ◽  
Surface Binding ◽  
High Molar Mass ◽  
Sugar Binding ◽  
Binding Pockets ◽  
Domain V

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.

scholarly journals Structure of chymopapain M the late-eluted chymopapain deduced by comparative modelling techniques and active-centre characteristics determined by pH-dependent kinetics of catalysis and reactions with time-dependent inhibitors: the Cys-25/His-159 ion-pair is insufficient for catalytic competence in both chymopapain M and papain

1994 ◽  
Vol 300 (3) ◽  
pp. 805-820 ◽  
Author(s):  
M P Thomas ◽  
C M Topham ◽  
D Kowlessur ◽  
G W Mellor ◽  
E W Thomas ◽  
...  
Keyword(s):  
Cation Exchange ◽  
Cysteine Proteinase ◽  
Ion Pair ◽  
Exchange Chromatography ◽  
Catalytic Site ◽  
Time Dependent ◽  
Cation Exchange Chromatography ◽  
Pka Values ◽  
Full Expression ◽  
Papaya Latex

Chymopapain M, the monothiol cysteine proteinase component of the chymopapain band eluted after chymopapains A and B in cation-exchange chromatography, was isolated from the dried latex of Carica papaya and characterized by kinetic and chromatographic analysis. This late-eluted chymopapain is probably a component of the cysteine proteinase fraction of papaya latex discovered by Schack [(1967) Compt. Rend. Trav. Lab. Carlsberg 36, 67-83], named papaya peptidase B by Lynn [(1979) Biochim. Biophys. Acta 569, 193-201] and partially characterized by Polgár [(1981) Biochim. Biophys. Acta 658, 262-269] and is the enzyme with unusual specificity characteristics (papaya proteinase IV) that Buttle, Kembhavi, Sharp, Shute, Rich and Barrett [Biochem. J. (1989) 261, 469-476] claimed to be a previously undetected cysteine proteinase eluted from a cation-exchange column near to the early-eluted chymopapains. A study of the time-dependent chromatographic consequences of thiol-dependent proteolysis of the components of papaya latex is reported. Chymopapain M was isolated by (i) affinity chromatography followed by separation from papain using cation-exchange f.p.l.c. on a Mono S HR5/5 column and (ii) cation-exchange chromatography followed by an unusual variant of covalent chromatography by thiol-disulphide interchange. The existence in chymopapain M of a nucleophilic interactive Cys/His catalytic-site system analogous to those in papain (EC 3.4.22.2) and other cysteine proteinases was deduced from the characteristics shape of the pH-second-order rate constant (k) profiles for its reactions with 2,2′-dipyridyl disulphide and ethyl 2-pyridyl disulphide. Analysis of the pH-k data for the reactions of chymopapain M with the 2-pyridyl disulphides and with 4,4′-dipyridyl disulphide permits the assignment of molecular pKa values of 3.4 and 8.7 to the formation and subsequent dehydronation of the Cys-S-/His-Im+H state of the catalytic site and reveals three other kinetically influential ionizations with pKa values 3.4, 4.3 and 5.6. The pH-dependences of kcat./Km for the hydrolysis of N-acetyl-L-Phe-Gly-4-nitroanilide at 25.0 degrees C and I0.1 M catalysed by chymopapain M and papain were determined. For both enzymes, little catalytic activity (5-7% of the maximal) develops consequent on formation of the catalytic site Cys-S-/His-Im+H ion-pair state (across pKa 3.4 for both enzymes). For papain, full expression of Kcat./Km for the uncharged substrate requires only the additional hydronic dissociation with pKa 4.2. By contrast, full expression of kcat./Km for chymopapain M requires additional hydronic dissociation with pKa values of 4.3 and 5.6.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Identification of signalling and non-signalling binding contributions to enzyme reactivity. Alternative combinations of binding interactions provide for change in transition-state geometry in reactions of papain

1989 ◽  
Vol 258 (3) ◽  
pp. 755-764 ◽  
Author(s):  
D Kowlessur ◽  
C M Topham ◽  
E W Thomas ◽  
M O'Driscoll ◽  
W Templeton ◽  
...  
Keyword(s):  
Transition State ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Thiol Group ◽  
Catalytic Site ◽  
Amide Bond ◽  
Compound I ◽  
Binding Interactions ◽  
Transition State Geometry ◽  
S2 Subsite

1. 2-(N'-Acetyl-L-phenylalanyl)hydroxyethyl 2′-pyridyl disulphide (compound V) was synthesized, and a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2) was used to evaluate the consequences for transition-state geometry of the presence of a hydrophobic occupant for the S2 subsite of the enzyme in the absence of the N-H component of the P1-P2 amide bond. 2. Comparison of the pH-dependences of K for reactions of compound (V), 2-(acetamido)ethyl 2′-pyridyl disulphide (compound I) and 2-(acetoxy)ethyl 2′-pyridyl disulphide (compound III) with the cysteine-proteinase minimal catalytic-site model, benzimidazol-2-ylmethanethiol, established the activation of all of these pyridyl disulphides by hydronation and that their reactivities are relatively insensitive to structural change in the non-pyridyl part of the molecule. The marked differences in their reactivities towards papain therefore derive from binding, either directly, or indirectly via signalling mechanisms. 3. Comparison of the kinetic data for the reaction of papain with compound (V) with those for analogous reactions with reactivity probes that provide opportunities for a variety of binding interactions in the S1-S2 intersubsite region and in the S2 subsite itself lead to the following conclusions: (a) the (Gly-66) N-H...O = C less than (P1-P2 ester) interaction of papain with compound (III) provides for better binding relative to that for a probe with a simple hydrocarbon side chain, but no signalling to the catalytic site to provide a (His-159)-ImH+-assisted transition state; (b) when this interaction is augmented either by a (P1-P2 amide) N-H...O = C less than (Asp-158) interaction (compound I) or hydrophobic P2/S2 contacts (compound V), signalling to the catalytic region occurs to provide the assisted transition state; (c) when both the P2/S2 contacts and the interaction involving Gly-66 exist, provision additionally of the (P1-P2 amide) N-H...O = C less than (Asp-158) interaction [as in 2-(N'-acetyl-L-phenylalanylamino)ethyl 2′-pyridyl disulphide] serves only to assist the binding without an additional signalling effect. 4. Such studies promise to allow binding interactions that merely locate substrates in appropriate enzyme loci to be distinguished from those that transmit signals with a chemical consequence to catalytic sites.

scholarly journals Organophosphate hydrolase interacts with ferric-enterobactin and promotes iron uptake in association with TonB dependent transport system.

2020 ◽  
Author(s):  
Hari Parapatla ◽  
Ramurthy Gudla ◽  
Guruprasad Varma Konduru ◽  
Elsin Raju Devadasu ◽  
Hampapathula Adimurthy Nagarajaram ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Iron Uptake ◽  
Docking Studies ◽  
Protein Docking ◽  
Catalytic Site ◽  
Organophosphate Hydrolase ◽  
E Coli ◽  
Target Membrane ◽  
Dependent Transport

Our previous studies have shown the existence of organophosphate hydrolase (OPH) as a part of the inner membrane associated TonB complex (ExbB/ExbD and TonB) of Sphingobium fuliginis. We now show its involvement in iron uptake by establishing direct interactions with ferric-enterobactin. The interactions between OPH and ferric-enterobactin were not affected even when the active site architecture is altered by substituting active site aspartate with either alanine or asparagine. Protein docking studies further substantiated these findings and predicted the existence of ferric-enterobactin binding site that is different from the catalytic site of OPH. A lysine residue (82 K) found at the predicted ferric-enterobactin binding site facilitated interactions between OPH and ferric-enterobactin. Substitution of lysine with alanine did not affect triesterase activity, but it abrogated OPH ability to interact with both ferric-enterobactin and ExbD, strengthening further the fact that the catalytic site is not the site for binding of these ligands. In the absence of interactions between OPHK82A and ExbD, OPHK82A failed to target membrane in E. coli cells. The Sphingobium fuliginis TonB dependent transport (SfTonBDT) system was reconstituted in E. coli GS027 cells generated by deleting the exbD and tonB genes. The E. coli GS030 cells having SfTonBDT system with OPH showed increased iron uptake. Such an increase was not seen in E. coli GS029, cells having SfTonBDT system generated either by omitting OPH or by including its variants, OPHD301A, OPHD301N suggesting a role for OPH in enhanced iron uptake.

scholarly journals A re-appraisal of the structural basis of stereochemical recognition in papain. Insensitivity of binding-site-catalytic-site signalling to P2-chirality in a time-dependent inhibition

1990 ◽  
Vol 266 (3) ◽  
pp. 645-651 ◽  
Author(s):  
W Templeton ◽  
D Kowlessur ◽  
E W Thomas ◽  
C M Topham ◽  
K Brocklehurst
Keyword(s):  
Transition State ◽  
Binding Site ◽  
Kinetic Data ◽  
Model Building ◽  
Ph Dependence ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Catalytic Site ◽  
Structural Basis ◽  
Compound I

1. 2-(N'-Acetyl-D-phenylalanylamino)ethyl 2′-pyridyl disulphide (compound I) [m.p. 123-124 degrees C; [alpha]20D -7.1 degrees (c 0.042 in methanol)] was synthesized, and the results of a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2), together with existing kinetic data for the analogous reaction of the L-enantiomer (compound II), were used to evaluate the consequences for transition-state geometry of the difference in chirality at the P2 position of the probe molecule. 2. The kinetic data suggest that the D-enantiomer binds approx. 40-fold less tightly to papain than the L-enantiomer but that the binding-site-catalytic-site signalling that results in a (His-159)-Im(+)-H-assisted transition state occurs equally effectively in the interaction of the former probe as in that of the latter. This results in pH-k profiles for the reactions of both enantiomers each characterized by four macroscopic pKa values (3.7-3.9, 4.1-4.3, 7.9-8.3 and 9.4-9.5) in which k is maximal at pH approx. 6 where the -Im(+)-H-assisted transition state is most fully developed. 3. Model building indicates that both enantiomers can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the binding pocket of the S2 subsite with preservation of the three hydrogen-bonding interactions involving the substrate analogue reagent and (Asp-158) C = O, (Gly-66) C = O, and (Gly-66)-N-H of papain. Earlier predictions that binding of N-acyl-D-phenylalanine derivatives to papain would be prevented on steric grounds [Berger & Schechter (1970) Philos. Trans. R. Soc. London B 257, 249-264; Lowe & Yuthavong (1971) Biochem. J. 124, 107-115; Lowe (1976) Tetrahedron 32, 291-302] were based on assumed models that are not consistent with the X-ray-diffraction data for papain inhibited by alkylation of Cys-25 with N-benzyloxycarbonyl-Phe-Ala-chloromethane [Drenth, Kalk & Swen (1976) Biochemistry 15, 3731-3738]. 4. The possibility that the kinetic expression of P2-S2 stereospecificity may depend on the nature of the chemistry occurring in the catalytic site of papain is discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

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