Kehinde O. Okonjo

Desensitization is a property of the cholinergic binding region of the nicotinic acetylcholine receptor, not of the receptor-integral ion channel.

The reversible acetylcholine esterase inhibitor (−)‐physostigmine (eserine) is the prototype of a new class of nicotinic acetylcholine receptor (nAChR) activating ligands: it induces cation fluxes into nAChR‐rich membrane vesicles from Torpedo marmorala electric tissue even under conditions of antagonist blocked acetylcholine binding sites (Okonjo, Kuhlmann, Maclicke, Neuron, in press). This suggests that eserine exerts its channel‐activating property via binding sites at the nAChR separate from those of the natural transmitter. We now report that eserine can activate the channel even when the receptor has been preincubated (desensitized) with elevated concentrations of acetylcholine. Thus the conformational state of the receptor corresponding to desensitization is confined to the transmitter binding region, leaving the channel fully activatable — albeit only from other than the transmitter binding site(s).

Biochemical Characterization of a Novel Channel-Activating Site on Nicotinic Acetylcholine Receptors

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine and several structurally related compounds with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles, direct binding studies and photoaffinity labeling. (-)Physostigmine acts as a channel-activating ligand at low concentrations and as a direct channel blocker at elevated concentrations. Channel activation is not inhibited by desensitizing concentrations of ACh or ACh-competitive ligands (including alpha-bungarotoxin and D-tubocurarine) but is inhibited by antibody FK1 and several other compounds. From photoaffinity labeling using tritiated physostigmine and mapping of the epitope for the Phy-competitive antibody FK1, the binding site for physostigmine is located within the alpha-subunit of the Torpedo nAChR and is distinct from the acetylcholine binding site. Our data suggest a second pathway of nAChR channel activation that may function physiologically as an allosteric control of receptor activity.

Temperature-jump studies on hemoglobin. Kinetic evidence for a non-quaternary isomerization process in deoxy- and carbonmonoxyhemoglobin.

Temperature jumps on mixtures of hemoglobin and pH indicators give rise to relaxation signals in the microsecond range. The pH and concentration dependences of the reciprocal relaxation time, 1/tau, may be rationalized on the basis of a reaction scheme in which a slow isomerization process in the protein moiety is coupled to a rapid co-operative ionization of two protons. At 11 degrees C the rate constants of the isomerization are kr = 4.2(+/- 1.8) x 10(4) s-1 and kf = 1.3(+/- 0.1) x 10(4) s-1 in deoxyhemoglobin; in carbonmonoxyhemoglobin they are kr = 3.9(+/- 1.3) x 10(4) s-1 and kf = 5.3(+/- 1.8) x 10(3) s-1. The pKa values of the coupled ionizing groups are 5.3 in deoxy- and 6.0 in carbonmonoxyhemoglobin. Modification of the CysF9(93) beta sulfhydryl group with iodoacetamide abolishes the pH dependence of 1/tau, suggesting that this sulfhydryl is involved in the isomerization process. Consideration of the X-ray structure of oxyhemoglobin allows a structural interpretation of the results. It is concluded that the isomerization may be important for the physiological function of hemoglobin, but that it is not identical with the quaternary structure transition.

Reactivities of the sulphydryl groups of dog hemoglobin

Abstract Dog hemoglobin has four sulphydryl groups at positions α111 (G18) and β93 (F9), all of which are titratable with mercurials. Only two of these, however, react with non-mercurial sulphydryl reagents. Kinetic results indicate that the reacting site might be the β93 (F9). An examination of the environment of the α111 (G18) shows that this sulphydryl must be unreactive towards non-mercurials because of the presence near it of several interacting groups. These are the carboxyl group of Glu 27α, which is only 4.5 Ă away; the carbonyl of Val 107α; and the hydroxyl of Tyr 24α. There is also a strong interaction with the carboxyl of Glu 116α which, though 12 A away, is separated from the α111 (G18) not by water but by protein, a low dielectric constant medium. All these interactions would considerably raise the pK of the Cys 111α thiol. Therefore reaction with non-mercurial sulphydryl reagents via nucleophilic attack by the thiol anion becomes impossible. The effect of inositol hexaphosphate on the kinetics of the sulphydryl group reaction was investigated. Inositol hexaphosphate slows down the reaction by a factor of three for a 10 M excess of inositol hexaphosphate per hemoglobin tetramer and makes about 25% of the sulphydryl contents of the β93 and α111 sites unavailable for reaction by any sulphydryl reagent.

Hemoglobins with multiple reactive sulphydryl groups : the reaction of dog hemoglobin with 5,5'-dithiobis (2-nitrobenzoate)

Dog hemoglobin has four sulphydryl groups per (tetramer) molecule located at the G18(111)a and F9(93)β positions. The two sulphydryls at the G18(111)a positions are unreactive toward nonmercurial sulphydryl reagents, but those at the F9(93)β positions are reactive toward these reagents. We have studied the kinetics of the reaction of dog hemoglobin with 5,5′-dithiobis (2-nitrobenzoic acid) as a function ofpH. At allpH values studied, the reaction is kinetically monophasic. Quantitative analysis of thepH dependence of the apparent second-order rate constant shows that two ionizable groups are linked to the reaction of the sulphydryl group. TheirpKa values are 5.57 and 9.0. These values are assigned to HisHC3(146)β and to the CysF9(93)β sulphydryl. We find that dog carbonmonoxyhemoglobin is significantly—almost an order of magnitude—less reactive than the aquomet, azidomet, and oxy derivatives. This result may be due to a greater tendency (at acidpH) for the salt bridge between HisHC3(146)β and AspFG1(94)β to form in the carbonmonoxy than in the other derivatives. Formation of this salt bridge is known to hinder access to the CysF9(93)β sulphydryl [Perutz, M. F. (1970),Nature228, 734–739].

Effect of A3[6]βGlu → Val mutation on reactivity of the CysF9[93]β sulfhydryl group of human haemoglobin S

The pH dependence profiles of the apparent second-order rate constant, kapp, for the reaction of the oxy, carbon monoxy and aquomet derivatives of human haemoglobin S with 5,5′-dithiobis(2-nitrobenzoate)(DTNB) in buffers of ionic strength 50 mmol dm–3 are complex. The pKas of the ionizable organic phosphate binding groups which influence the reactivity of the CysF9[93]β sulfhydryl group have been determined from quantitative analyses of the complex profiles. These pKa values were not significantly different from those of haemoglobin A. In the presence of inositol hexakisphosphate (inositol-P6) each profile assumes a simple form resembling the titration curve of a diprotic acid. The pKas of HisHC3[146]β and CysF9[93]β were determined from quantitative analyses of the simple profiles. The mean values obtained, 6.6 ± 0.2 and 8.84 ± 0.04, respectively, were the same as those of haemoglobin A. Comparison of the kapp data for haemoglobin S with those of haemoglobin A shows that, irrespective of the presence or absence of inositol-P6 presence, the carbon monoxy and aquomet derivatives of haemoglobin A react more rapidly than the corresponding haemoglobin S derivatives. In contrast, in the absence of inositol-P6, oxyhaemoglobin A reacts faster than oxyhaemoglobin S; in the presence of the organic phosphate both haemoglobins react at about the same rate. At an ionic strength of 200 mmol dm–3 in the absence of inositol-P6, the pH dependence profiles of kapp for the oxy, carbon monoxy and aquomet derivatives of haemoglobins A and S are simple. Quantitative analyses of these profiles give mean pKa values of 5.4 ± 0.1 and 8.9 ± 0.2 for HisHC3[146]β and CysF[93]β, respectively. The oxy and carbon monoxy derivatives of both haemo-globins react at about the same rate, but aquomethaemoglobin A reacts significantly more rapidly than aquomethaemoglobin S.

Organic phosphate-binding groups electrostatically linked to the reactivity of the Cys F9 [93]β sulfhydryl group of haemoglobin

At an ionic strength of 0.05 mol dm–3 the pH-dependence profile for the reaction of the Cys F9 [93]β sulfhydryl group of human haemoglobin (stripped of organic phosphates) with 5,5′-dithiobis(2-nitrobenzoate)(DTNB) is complex. In the presence of a four-fold molar excess of inositol hexakisphosphate (inositol-P6) over haemoglobin tetramers, the pH-dependence profile changes dramatically from a complex to a simple form resembling the titration curve of a diprotic acid. Values of the apparent second-order rate constant, kapp, are also drastically reduced. Quantitative analyses of the simple profiles indicate that the reactivity of the sulfhydryl is linked to the ionization of two amino acid residues on the protein, with pKa values around 6.6 and 8.7. These pKas are assigned to His HC3 [146]β and Cys F9 [93]β, respectively.Since inositol-P6 simplifies the complex pH-dependence profile obtained for stripped haemoglobin by binding to the cationic groups at the organic phosphate binding site, we have analysed the complex pH-dependence profile of stripped haemoglobin quantitatively by assuming that there is an electrostatic interaction between the sulfhydryl and the cationic groups. From the analyses of the complex profiles for the oxy, carbon monoxy, azidomet and cyanomet derivatives, mean pKa values of 6.4 ± 0.1, 7.5 ± 0.2 and 9.5 ± 0.02 are obtained for the groups that are electrostatically linked to the Cys F9 [93]β sulfhydryl group. The pKa of 6.4 is assigned to His NA2 [2]β and HisH21 [143]β; the pKa of 7.5 is assigned to Val NA1 [1]β; and the pKa of 9.5 is assigned to Cys F9 [93]β. For aquomethaemoglobin, analysis shows that, in addition to these groups, the water molecule attached to the sixth coordination position of the iron(III) atom is also electrostatically linked to the sulfhydryl.

Ligand-dependent reactivity of the CysB5[23] β sulfhydryl group of the major haemoglobin of chicken

Chicken haemoglobin contains eight reactive sulfhydryl groups per (tetramer) molecule, as determined by Boyer titration with p-chloromercury(II)benzoic acid. However, only four of these sulfhydryls are reactive towards 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). They are at the F9[93] and B5[23] positions of each of the two β subunits in the molecule. The time course of the DTNB reaction is biphasic. With oxyhaemoglobin, k app , the apparent second-order rate constant of the fast phase, increases monotonically with pH, the simple profile resembling the titration curve of a diprotic acid; the pH-dependence of k app for the slow phase is bowl-shaped. With carbonmonoxyhaemoglobin and aquomethaemoglobin, k app for the fast phase is bowl-shaped whilst k app for the slow phase increases monotonically with pH. Quantitative analyses of the simple profiles show that the reactivity of the sulfhydryl group to which they may be attributed is subject to the influence of two ionizable groups on the molecule, with mean pK a values of 6.4±0.1 and ca. 8.4±0.3. These pK a values are assigned to HisHC3[146]β and CysF9[93]β, respectively. Quantitative analyses of the bowl-shaped profiles show that the reactivity of the sulfhydryl group to which they may be attributed is subject to the influence of two ionizable groups on the protein, with mean pK a s of 6.85±0.05 and 8.3±0.2. These values are assigned to HisG19[117]β and CysB5[23]β, respectively. It is highly significant that the CysB5[23]β sulfhydryl groups of carbonmonoxy- and aquomet-haemoglobin react ca. 100 times faster than that of oxyhaemoglobin. By contrast, the difference in the reactivities of the CysF9[93]β sulfhydryls of the three haemoglobin derivatives is no more than four-fold. This indicates that, in chicken haemoglobin, changes in the haem ligand give rise to structural changes in the neighbourhood of the CysB5[23]β sulfhydryl which are far more significant than those in the neighbourhood of the CysF9[93]β sulfhydryl.

Bohr effect of avian hemoglobins: Quantitative analyses based on the Wyman equation

The Bohr effect data for bar-headed goose, greylag goose and pheasant hemoglobins can be fitted with the Wyman equation for the Bohr effect, but under one proviso: that the pKa of His146β does not change following the T→R quaternary transition. This assumption is based on the x-ray structure of bar-headed goose hemoglobin, which shows that the salt-bridge formed between His146β and Asp94β in human deoxyhemoglobin is not formed in goose deoxyhemoglobin. When the Bohr data for chicken hemoglobin were fitted by making the same assumption, the pKa of the NH3+ terminal group of Val1α decreased from 7.76 to 6.48 following the T→R transition. When the data were fitted without making any assumption, the pKa of the NH3+ terminal group increased from 7.57 to 7.77 following the T→R transition. We demonstrate that avian hemoglobin Bohr data are readily fitted with the Wyman equation because avian hemoglobins lack His77β. From curve-fitting to Bohr data we estimate the pKas of the NH3+ terminal group of Val1α in the R and T states to be 6.33±0.1 and 7.22±0.1, respectively. We provide evidence indicating that these pKas are more accurate than estimates from kinetic studies.

Subunit iron spin heterogeneity in human aquomethemoglobin A

On the basis of a reaction scheme in which the ligand binding steps are preceded by fast iron spin transitions (Okonjo, K.O. (1980) Eur. J. Biochem. 105, 329-334; Iwuoha, E.I. and Okonjo, K.O. (1985) Biochim. Biophys. Acta 829, 327-334), the spin equilibrium constants of methemoglobin subunits are calculated from kinetic and equilibrium binding parameters with azide ion as ligand. The results demonstrate the existence of thermodynamic spin heterogeneity within the tetramer.