The homodimeric hemoglobin from Scapharca can be locked into new cooperative structures upon reaction of Cys92, located at the subunit interface, with organomercurials. (1/6)

In the cooperative, homodimeric hemoglobin from Scapharca inaequivalvis, HbI, the subunit interface is formed by the heme-carrying E and F helices and contains the only cysteine residue of the globin chain (Cys92, F2) in an area which changes from hydrophilic to hydrophobic upon oxygenation. Binding of organomercurials to HbI is cooperative and entails major quaternary rearrangements. The reaction of Cys92 with p-chloromercuri-benzoate (PMB) and p-nitro-o-chloromercuriphenol (PN), a sensitive reporter of the cysteine microenvironment at neutral pH values, has been followed in stopped flow experiments. Kinetic evidence for the cooperativity of mercurial binding has been obtained and the rate of the corresponding conformational transition has been estimated. As expected PN, but not PMB, is able to monitor the oxygen-linked change of the cysteine microenvironment. The modification of Cys92 with PN has unique functional effects. In PN-reacted HbI cooperativity is maintained, albeit to a different extent, depending on the ligation state of the protein during mercaptide formation. It may be envisaged that PN locks the protein into new, cooperative, quaternary structures stabilized by hydrogen bonding interactions between the ionized nitrophenol moiety and the contralateral subunit.  (+info)

Kinetic differentiation between enzyme inactivation involving complex-formation with the inactivator and that involving a conformation-change step. (2/6)

It has been suggested that the complexing type of inactivation in which the inactivator binds reversibly with the enzyme before inactivation cannot be differentiated kinetically from that a slow enzyme conformation change is involved as a first step [Rakitzis (1986) J. Theor. Biol. 122, 247-249]. The kinetics of the substrate reaction during modification of enzyme activity previously described [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381-436] have now been applied to this problem and equations derived to show that the slow-conformational-change type can be differentiated from the complexing type by plotting the final concentration of product formed, [P]infinity, against the reciprocal of inactivator concentration. The reaction of hexokinase with 2-chloromercuri-4-nitrophenol has been shown to involve a conformational change of the enzyme before inactivation.  (+info)

Use of isoelectric focusing and a chromophoric organomercurial to monitor urea-induced conformational changes of yeast phosphoglycerate kinase. (3/6)

The effects of urea in concentrations from 0 to 6M on the following properties of yeast phosphoglycerate kinase were studied: the kinetics of inactivation of the enzyme, the spectrum of 2-chloromercuri-4-nitrophenol bound to the single thiol group of the enzyme, the rate of reaction between the mercurial and enzyme, and the isoelectric point. The enzyme was inactivated by as much as 30% in 1M-urea, and the other data were interpreted as a possible 'tightening' of enzyme structure. The catalytic behaviour of the enzyme in 2M-urea was time-dependent, the initial effects being similar to those in 1M-urea. Polyacrylamide-gel isoelectric focusing of the enzyme in the presence of 2M-urea showed a single species of enzyme with an isoelectric point intermediate between those in 1M- and 3M-urea; a species with an identical isoelectric point was obtained after an 11-day exposure at 4 degrees C to the denaturant at 2M. The enzyme was rapidly inactivated in 3M-urea, with the thiol group fully exposed and the isoelectric point 0.9pH unit higher than in the absence of urea. No further conformational changes could be demonstrated with urea concentrations of 4M or greater. It is suggested that the equilibrium species that exists in 2M-urea has one of two buried lysine residues exposed. The second lysine residue is exposed in 3M or greater concentrations of the denaturant.  (+info)

A thiol-labelling reagent and reactivity probe containing electrophilic mercury and a chromophoric leaving group. (4/6)

2-(2'-Pyridylmercapto)mercuri-4-nitrophenol was synthesized and evaluated as a thiol-labelling reagent containing a chromophoric leaving group and as a reactivity probe by studies on its reactions with 2-mercaptoethanol and with papain (EC 3.4.22.2).  (+info)

Human 'creatine kinase conversion factor' identified as a carboxypeptidase. (5/6)

The effect of partially purified 'creatine kinase conversion factor' on rabbit muscle creatine kinase is shown to be that of a carboxypeptidase, removing the C-terminal lysine residue from both subunits. These changes fully explain the three-banded electrophoretic patterns of the partially and the fully modified rabbit and human enzymes. The factor also produces a similar electrophoretic pattern with haemoglobin A; comparison with the effects of carboxypeptidases A and B permits the inference that the C-terminal residues of both alpha- and beta-subunits are removed. Small synthetic peptides are poor or non-substrates. A low activity with hippuryl-L-lysine may be due to contamination of the preparation with carboxypeptidase N. The possibility has been excluded that the action of conversion factor on creatine kinase involves modification of the protein thiol groups. Mr, substrate-specificity, pH-activity profile and the effects of metal ions distinguish creatine kinase conversion factor from carboxypeptidases A, B and N. On the basis of this evidence it is proposed to give the conversion factor the provisional name of carboxypeptidase K.  (+info)

The essential sulfhydryl group of ornithine transcarbamylases. pH dependence of the spectra of its 2-mercuri-4-nitrophenol derivative. (6/6)

The essential sulfhydryl group of the ornithine transcarbamylases (ornithine carbamoyltransferase, 2.1.3.3) from bovine liver and Streptococcus faecalis reacts preferentially with 2-chloromercuri-4-nitrophenol. The spectra of this derivative between pH 4.4 AND 8.8 HAVE BEEN RESOLVED INto the spectrum of the nitrophenolate ion (III) and two species of phenol (I and II). The lambda max of I and II (both enzymes) and III (bovine) are red shifted from those of the comparable species in the same derivative of 2-mercaptoethanol. Deprotonation of a residue on the enzyme must be responsible for the transition from I to II. The pK values of the phenolic group are 7.1 (mercaptoethanol), 7.7 (bovine), and 8.8 (S. faecalis). The red shift in the lambda max of III and the modest increase in the pK of the phenolic group are consistent with a relatively hydrophobic environment for the nitrophenolate ion in the bovine enzyme. Since deprotonation of the residue in the bovine enzyme perturbs the pK of the phenolic group only slightly, its effect may be indirect. Interaction with a neighboring carboxyl group (pK 5.3) would account for the large increase in the pK of the phenolic group in the S. faecalis enzyme, which is not accompanied by an appreciable shift in the lambda max. Carbamyl-P increases the pK of the phenolic group in both enzymes, a result consistent with its binding site being close to the essential sulfhydryl group.  (+info)