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E W Thomas

Publications and source records attributed to E W Thomas.

At least 37 records · Page 2Linked to original sources
PubMed1993

Clarification of the pH-dependent kinetic behaviour of papain by using reactivity probes and analysis of alkylation and catalysed acylation reactions in terms of multihydronic state models: implications for electrostatics calculations and interpretation of the consequences of site-specific mutations such as Asp-158-Asn and Asp-158-Glu.

1. The complex behaviour of papain (EC 3.4.22.2) in acidic media has been investigated by (a) stopped-flow reactivity probe kinetics using 4,4'-dipyrimidyl disulphide (I) and 2,2'-dipyridyl disulphide (II) as thiol-specific time-dependent inhibitors with markedly different susceptibilities to activation by hydronation (protonation) and (b) using the multitasking application program SKETCHER for the rapid evaluation of pH-dependent kinetic data by means of interactive manipulation of calculated curves. 2. The substantially lower basicity of (I) (pKa 0.91) than that of (II) (pKa 2.45) combined with retention of high reactivity permitted the pKa for the formation of the (Cys-25)-S-/(His-159)-Im+H ion-pair state of papain to be determined kinetically as 3.4, a value close to that (3.3) deduced by potentiometric difference titration [Lewis, Johnson and Shafer (1976) Biochemistry 15, 5009-5017] and lower than the value (approx. 4) often reported from pH-dependent kinetic studies. The higher values are now known to arise from inadequate data analysis that does not take account of other overlapping kinetically influential ionizations. 3. Re-evaluation of the extensive sets of pH-kcat/Km data for the hydrolysis of nine substrates by papain reported by Polgár and Halász (1978) (Eur. J. Biochem. 88, 513-521) by making use of SKETCHER, the known pKa value (3.4) from the reaction with compound (I) and two additional kinetically influential pKa values deduced from the reaction with compound (II) now permits the identification of the pH-dependent events in reactions of papain with inhibitors and substrates. 4. A major conclusion is that, whereas in reactions of simple alkylating agents and compound (I) full nucleophilic character of (Cys-25)-S-/(His-159)-Im+H is provided by hydronic dissociation with pKa 3.3-3.4, in catalysis relatively little catalytic competence is produced consequent upon ion-pair formation. Substantial catalytic competence requires further hydronic dissociation with pKa approx. 4, and for cationic substrates further enhancement is produced by hydronic dissociation with pKa approx. 5. 5. The present work, together with the kinetic analysis of reactions of papain in alkaline media reported by Mellor, Thomas, Topham and Brocklehurst [Biochem. J. (1993) 290, 289-296], defines the kinetically influential ionizations of papain as 3.4, 4.0, 5.0, 8.3 and 10.0 of which 3.4 and 8.3 relate to the formation and subsequent dehydronation of the ion-pair state.(ABSTRACT TRUNCATED AT 400 WORDS)

Acylation↗
PubMed1993

Catalytic-site characteristics of the porcine calpain II 80 kDa/18 kDa heterodimer revealed by selective reaction of its essential thiol group with two-hydronic-state time-dependent inhibitors: evidence for a catalytic site Cys/His interactive system and an ionizing modulatory group.

1. Four calpain II heterodimers (80 kDa/30 kDa, 80 kDa/29 kDa, 80 kDa/26 kDa and 80 kDa/18 kDa) were isolated from fresh porcine kidney by (NH4)2SO4 precipitation, chromatography on DEAE-Sepharose CL-6B and subsequently on Reactive Red 120/agarose followed by f.p.l.c. on a Q-Sepharose Hi-Load 16/10 column. 2. The major component (80 kDa/30 kDa) was used to provide the catalytically active calpain II 80 kDa/18 kDa heterodimer by treatment with CaCl2; titration with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64) in the presence of monothioglycerol showed the preparation to have 1.0 +/- 0.05 catalytic sites per molecule of heterodimer. 3. The 80 kDa/30 kDa heterodimer was separated from monothioglycerol and other low-molecular-mass material by gel filtration on Sephadex G-25 without loss of catalytic activity towards sulphanilic acid/azocasein in the presence of added Ca2+. On storage overnight at a concentration of 3 microM in KCl at 4 degrees C in the absence of Ca2+ the activator-free preparation still produced fully active 80 kDa/18 kDa heterodimer on addition of Ca2+. 4. Activator-free 80 kDa/30 kDa heterodimer (in the absence of Ca2+) reacts relatively slowly with ethyl 2-pyridyl disulphide at pH 5.9; over 5000 s five thiol groups per molecule react, all at similar rates. In the presence of 8 mM CaCl2 under otherwise identical conditions (and also in the pH range 3.8-10.4) an initial faster phase of reaction corresponding to approx. one thiol group per molecule of heterodimer is generated, but it is not cleanly separated from the subsequent slower reactions on the stopped-flow trace. This fast phase of reaction does not occur when E64-inactivated calpain II is substituted for active 80 kDa/18 kDa heterodimer. 5. Greatly improved resolution of the fast phase of reaction involving the catalytic-site thiol group was achieved by using 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) instead of ethyl 2-pyridyl disulphide. 6. The pH-dependence of the second-order rate constant (k) for the reaction of the catalytically active activator-free 80 kDa/18 kDa calpain II heterodimer with 2-Py-S-S-2-Py was studied by stopped-flow spectral analysis in the pH range approx. 3-8 without interference from reactions of other thiol groups. 7. The form of the pH-k profile establishes for the first time the existence of an interactive catalytic site system [probably containing a (Cys)-S-/(His)-Im+H ion pair] analogous to those present in monomeric non-Ca(2+)-activated cysteine proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Ionization characteristics of the Cys-25/His-159 interactive system and of the modulatory group of papain: resolution of ambiguity by electronic perturbation of the quasi-2-mercaptopyridine leaving group in a new pyrimidyl disulphide reactivity probe.

1. A new thiol-specific reactivity probe 4,4'-dipyrimidyl disulphide [compound (VII), m.p. 110 degrees C, pKa of its monohydronated form 0.91] was synthesized and used to resolve the ambiguity of interpretation of the behaviour of papain (EC 3.4.22.2) in alkaline media known to depend to varying extents on two ionizations with pKa values approx. 8.0-8.5 and > or = 9.5 respectively. 2. A new extensive pH-second-order rate constant (k) data set for the reaction of papain with 2-(acetamido)-ethyl 2'-pyridyl disulphide (IV) demonstrated the existence of a striking rate maximum at pH approx. 4, the independence of k around pH 8 and the increase in k with increase in pH across a pKa value of 10.0, behaviour similar to that of other 2-pyridyl disulphides (R-S-S-2-Py) that lack key substrate-like binding sites in R. 3. Although the simplest interpretation of the pKa value of 10.0 assigns it to the formation of (Cys-25)-S-/(His-159)-Im from the ion-pair state of the papain catalytic site, another interpretation may be conceived in which this pKa value is assigned to another group remote from the catalytic site, the state of ionization of which modulates catalytic-site behaviour. This alternative assignment is shown to require compensating effects in the pH region around 8 such that the formation of (Cys-25)-S-/(His-159)-Im across pKa 8.0-8.5 is without net kinetic effect in the reactions of simple 2-pyridyl disulphides such as compound (IV) and 2,2'-dipyridyl disulphide (II). 4. The lower basicity of compound (VII) relative to that of compound (II) (pKa 2.45) was predicted to diminish or abolish the compensation postulated as a possibility in reactions of 2-pyridyl disulphides because of the decreased effectiveness of reaction via a (His-159)-Im+H-assisted transition state. The characteristics of the pH-dependence of the reaction of papain with compound (VII) which are quite different from those for its reaction with compound (II) support both this prediction and the alternative assignment with a value of 8.3 for the pKa of the formation of (Cys-25)-S-/(His-159)-Im. 5. Evidence that the behaviour of papain towards both substrates and some substrate-derived time-dependent inhibitors is determined not only by the loss of the (Cys-25)-S-/(His-159)-Im+H ion-pair state by dehydronation with pKa 8.3 but also by another ionization of pKa approx. 10.0 is briefly discussed.

Binding Sites↗
PubMed1992

Evaluation of hydrogen-bonding and enantiomeric P2-S2 hydrophobic contacts in dynamic aspects of molecular recognition by papain.

1. 2-(N'-Acetyl-D-phenylalanyl)hydroxyethyl 2'-pyridyl disulphide (compound IV) (m.p. 59 degrees C; [alpha]D20 -6.6 degrees (c 1.2 in methanol)) was synthesized. 2. 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 (Cys-25) of papain (EC 3.4.22.2) together with analogous kinetic data for the reactions of related time-dependent inhibitors, notably the L-enantiomer of compound (IV) (compound III) and the L- and D-enantiomers of 2-(N'-acetylphenylalanylamino)ethyl 2'-pyridyl disulphide (compounds I and II respectively), were used to assess the contributions of the (P1)-NH ... O = C < (Asp-158) and (P2) > C = O ... H-N-(Gly-66) hydrogen bonds and enantiomeric P2-S2 hydrophobic contacts in two manifestations of dynamic molecular recognition in papain-ligand association: (a) signalling to the catalytic-site region to provide for a (His-159)-IM(+)-H-assisted transition state and (b) the dependence of P2-S2 stereoselectivity on hydrogen-bonding interactions outside the S2 subsite. The analysis involved determination of the reactivities of individual ionization states of the reactions (pH-independent rate constants, k) and associated macroscopic pKa values and difference kinetic specificity energies (delta delta GKS = -RT1n(k1/k2), where k1 is the pH-independent second-order rate constant for reaction with one inhibitor and k2 is the analogous rate constant in the same ionization state for reaction with another inhibitor so that, when the structural change provides that k2 > k1, delta delta GKS is positive. 3. The kinetic data further illuminate the nature of the interdependence of binding interactions in papain first noted by Kowlessur, Topham, Thomas, O'Driscoll, Templeton & Brocklehurst [(1989) Biochem. J. 258, 755-764] in the S2 subsite, S1-S2 intersubsite and catalytic-site regions. Of particular note is the apparent dependence of the binding of the N-Ac-D-Phe moiety on the binding of the leaving group to (His-159)-Im+H and the fact that the resulting rate enhancement is more effective when (P1)-N-H is absent than when it is present. This result revealed by kinetic analysis goes beyond the conclusion suggested by model building that it is possible to make all of the binding contacts in complexes involving the D-enantiomers [(II) and (IV)] as in those involving the L-enantiomers [(I) and (III)].(ABSTRACT TRUNCATED AT 400 WORDS)

Catalysis↗
PubMed1992

Cholesterol lowering bile acid binding agents: novel lipophilic polyamines.

A series of novel lipophilic polyamines was synthesized by the sodium cyanoborohydride-mediated reductive amination of various ketones and aldehydes with the polyamine tris(2-aminoethyl)amine. Two of these compounds, N,N-bis[2-(cyclododecylamino)ethyl]-N'-benzyl-1,2-ethanediamine trihydrochloride (36.3HCl) and N,N-bis[2-(cyclododecylmethylamino)ethyl]-N',N'-dimethyl-1,2-ethan ediamine (23), are 29 and 24 times more potent than colestipol hydrochloride, respectively, for lowering animal serum cholesterol levels.

Animals↗
PubMed1992

Variation in the P2-S2 stereochemical selectivity towards the enantiomeric N-acetylphenylalanylglycine 4-nitroanilides among the cysteine proteinases papain, ficin and actinidin.

1. Values of the kinetic specificity constant, kcat./Km, for the hydrolysis of N-acetyl-L-phenylalanylglycine 4-nitroanilide (I) and of its D-enantiomer (II) catalysed by ficin (EC 3.4.22.3) and by actinidin (EC 3.4.22.14) at pH 6.0, I 0.1 mol/l, 8.3% (v/v) NN-dimethylformamide and 25 degrees C were determined by using initial-rate data with [S] much less than Km and weighted nonlinear regression analysis as: for ficin, (kcat./Km)L = 271 +/- 6 M-1.s-1, (kcat./Km)D = 2.9 +/- 0.1 M-1.s-1, and for actinidin (kcat./Km)L = 13.3 +/- 0.7 M-1.s-1, (kcat/Km)D = 0.34 +/- 0.01 M-1.s-1.2. These data and analogous values for the corresponding reactions catalysed by papain (EC 3.4.22.2), (kcat./Km)L = 2064 +/- 31 M-1.s-1, (kcat./Km)D = 5.5 +/- 0.1 M-1.s-1, demonstrate marked variation in stereochemical selectivity for substrates (I) and (II) among the three cysteine proteinases with the following values for the index of stereochemical selectivity Iss = (kcat./Km)L/(kcat./Km)D: for papain, 375; for ficin 93; for actinidin 39. 3. Model building suggests ways in which, for the papain-catalysed reactions, binding interactions involving the extended acyl groups of the substrates may need to change as the reaction proceeds from adsorptive complex (ES) to tetrahedral intermediate (THI) before its rate-determining, general acid-catalysed collapse to acylenzyme intermediate. In particular, satisfactory alignment in the catalytic site at the THI stage of the acylation process appears to demand rotation of the substrate moiety about its long axis. 4. The different consequences of this rotation for the L- and D-enantiomers suggest that for closely related systems the greater the extent of this rotational adjustment the greater would be the value of Iss.5. For the actinidin-substrate combinations, model building suggests that even at the ES complex stage of catalysis it is not possible to approach optimized P2-S2 contacts and the three hydrogen-bonding interactions deduced for papain-ligand complexes in the absence of significant movement of protein conformation. Possible binding modes in which some of the interactions deduced for papain are relaxed are discussed. Consideration of postulated binding modes in the various transition states is shown to account for the order of reactivity reflected in values kcat./Km for the four reactions involving papain (Pap) and actinidin (Act) with the L- and D-enantiomeric substrates: Pap-L much greater than Act-L greater than Pap-D much greater than Act-D.(ABSTRACT TRUNCATED AT 400 WORDS)

Anilides↗
PubMed1991

Structure-function relationships in the cysteine proteinases actinidin, papain and papaya proteinase omega. Three-dimensional structure of papaya proteinase omega deduced by knowledge-based modelling and active-centre characteristics determined by two-hydronic-state reactivity probe kinetics and kinetics of catalysis.

1. A model of the three-dimensional structure of papaya proteinase omega, the most basic cysteine proteinase component of the latex of papaya (Carica papaya), was built from its amino acid sequence and the two currently known high-resolution crystal structures of the homologous enzymes papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14). The method used a knowledge-based approach incorporated in the COMPOSER suite of programs and refinement by using the interactive graphics program FRODO on an Evans and Sutherland PS 390 and by energy minimization using the GROMOS program library. 2. Functional similarities and differences between the three cysteine proteinases revealed by analysis of pH-dependent kinetics of the acylation process of the catalytic act and of the reactions of the enzyme catalytic sites with substrate-derived 2-pyridyl disulphides as two-hydronic-state reactivity probes are reported and discussed in terms of the knowledge-based model. 3. To facilitate analysis of complex pH-dependent kinetic data, a multitasking application program (SKETCHER) for parameter estimation by interactive manipulation of calculated curves and a simple method of writing down pH-dependent kinetic equations for reactions involving any number of reactive hydronic states by using information matrices were developed. 4. Papaya proteinase omega differs from the other two enzymes in the ionization characteristics of the common (Cys)-SH/(His)-Im+H catalytic-site system and of the other acid/base groups that modulate thiol reactivity towards substrate-derived inhibitors and the acylation process of the catalytic act. The most marked difference in the Cys/His system is that the pKa for the loss of the ion-pair state to form -S-/-Im is 8.1-8.3 for papaya proteinase omega, whereas it is 9.5 for both actinidin and papain. Papaya proteinase omega is similar to actinidin in that it lacks the second catalytically influential group with pKa approx. 4 present in papain and possesses a catalytically influential group with pKa 5.5-6.0. 5. Papaya proteinase omega occupies an intermediate position between actinidin and papain in the sensitivity with which hydrophobic interaction in the S2 subsite is transmitted to produce changes in transition-state geometry in the catalytic site, a fact that may be linked with differences in specificity in P2-S2 interaction exhibited by the three enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

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.

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)

Binding Sites↗
PubMed1990

Dependence of the P2-S2 stereochemical selectivity of papain on the nature of the catalytic-site chemistry. Quantification of selectivity in the catalysed hydrolysis of the enantiomeric N-acetylphenylalanylglycine 4-nitroanilides.

1. N-Acetyl-L-phenylalanylglycine 4-nitroanilide and its D-enantiomer were synthesized and characterized and used as substrates with which to evaluate stereochemical selectivity in papain (EC 3.4.22.2)-catalysed hydrolysis. 2. Kinetic analysis at pH 6.0, I 0.1, 8.3% (v/v) NN-dimethylformamide and 25 degrees C by using initial-rate data with [S] much less than Km and weighted non-linear regression provided values of kcat./Km for the catalysed hydrolysis of both enantiomers as (kcat./Km)L = 2040 +/- 48 M-1.S-1 and (kcat./Km)D = 5.9 +/- 0.07 M-1.S-1. These data, taken together with individual values of kcat. and Km for the hydrolysis of the L-enantiomer (a) estimated in the present work as kcat. = 3.2 +/- 1.2 S-1 and Km = 1.5 +/- 0.6 mM and (b) reported by Lowe & Yuthavong [(1971) Biochem. J. 124, 107-115] for the reaction at pH 6.0 in 10% (v/v) NN-dimethylformamide and 35 degrees C, as kcat. = 1.3 +/- 0.2 S-1 and Km = 0.88 +/- 0.1 mM, suggest that (kcat./Km)L congruent to 2000 M-1.S-1 and thus that (kcat./Km)L/(kcat./Km)D congruent to 330.3. Model building indicates that both enantiomeric 4-nitroanilides can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the S2 subsite with preservation of mechanistically relevant hydrogen-bonding interactions and that the main difference is in the positioning of the beta-methylene group. 4. The dependence of P2-S2 stereochemical selectivity of papain on the nature of the catalytic-site chemistry for reactions involving derivatives of N-acetylphenylalanine is discussed. The variation in the index of stereochemical selectivity (ratio of the appropriate kinetic or thermodynamic parameter for a given pair of enantiomeric ligands), from 330 for the overall acylation process of the catalytic act, through 40 and 31 for the reaction at electrophilic sulphur in 2-pyridyl disulphides respectively without and with assistance by (His-159)-Im(+)-H, to 5 for the formation of thiohemiacetal adducts by reaction at aldehydic carbon, is interpreted in terms of the extent to which conformational variation of the bound ligand in the catalytic-site region permits the binding mode of the -CH2-Ph group of the D-enantiomer to approach that of the L-enantiomer.

Binding Sites↗
PubMed1989

The interplay of electrostatic fields and binding interactions determining catalytic-site reactivity in actinidin. A possible origin of differences in the behaviour of actinidin and papain.

1. The pH-dependence of the second-order rate constant (k) for the reaction of actinidin (EC 3.4.22.14) with 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide was determined and the contributions to k of various hydronic states were evaluated. 2. The data were used to assess the consequences for transition-state geometry of providing P2/S2 hydrophobic contacts in addition to hydrogen-bonding opportunities in the S1-S2 intersubsite region. 3. The P2/S2 contacts (a) substantially improve enzyme-ligand binding, (b) greatly enhance the contribution to reactivity of the hydronic state bounded by pKa 3 (the pKa characteristic of the formation of catalytic-site-S-/-ImH+ state) and pKa 5 (a relatively minor contributor in reactions that lack the P2/S2 contacts), such that the major rate optimum occurs at pH 4 instead of at pH 2.8-2.9, and (c) reveal the kinetic influence of a pKa approx. 6.3 not hitherto observed in reactions of actinidin. 4. Possibilities for the interplay of electrostatic effects and binding interactions in both actinidin and papain (EC 3.4.22.2) are discussed.

Binding Sites↗
PubMed1989

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.

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.

Animals↗