Proton magnetic resonance titration curves of the three histidine residues of staphylococcal protease

Abstract

Proton magnetic resonance spectra of staphylococcal protease, a serine protease from Staphylococcus aureus, strain V8, are presented. Initial proton spectra were obtained at 220 MHz, and more detailed studies of the aromatic region were carried out by correlation spectroscopy at 250 MHz. The overall spectrum bears a close resemblance to one calculated from the sum of spectra of the component amino acids. Chemical shifts of the three tyrosine, four phenylalanine, and three histidine residues appear to be equivalent at pH 3.7 and 8.5 indicating that they are all in normal chemical environments in the enzyme. The staphylococcal protease contains a large number of slowly exchanging protons. In fact, interpretable spectra of the aromatic region were obtained only after extensive exchange of N-H groups with deuterium from the D2O solvent. Proton magnetic resonance titration studies of the three histidine residues indicate that these have normal chemical shifts and pK' values. When the data are fitted to single noninteracting titration curves, the histidine pK' values are 7.19 plus or minus 0.02, 6.85 plus or minus 0.03, and 6.69 plus or minus 0.02. The titration curves of two of the histidine residues indicate negative cooperativity. A possible explanation for this is a direct electrostatic interaction between the two histidines. The titration data for these histidines give a significantly better fit to such a mutual interaction model than to noninteracting titration curves. The component microscopic dissociation constants have been calculated. Mutual interaction leads to pK' displacements of 0.31 unit; which indicates a distance of approximately 7 angstrom between the two interacting histidine rings according to the model of Tanford and Roxby. The proton resonances of the two interacting histidines are doubled in the pH region 6.7-7.0 suggesting the presence of two forms of the enzyme having lifetimes in excess of 30 msec.

Transition-state analysis of the facilitated alkylation of ribonuclease A by bromoacetate

“…The p H dependency for binding an anion, such as shown for bromoacetate in Figure 2, means that the effects will be pH dependent unless the concentration of anion is saturating at all p H values studied. The pK values found from carboxymethylation of RNase are close to values for imidazole and its derivatives and to the pK values determined by N M R titration: His-119, pK 6.2; His-12, pK 5.8, in D 2 0 and in the presence of 0.3 M NaCl (Markley & Finkenstadt, 1975). Thus, the enhanced reactivities of the histidine residues in RNase do not seem to be accompanied by large perturbations in pK values.…”

“…Investigations of RNase often employ sodium acetate or sodium chloride to control pH or adjust ionic strength of solutions of the enzyme. In particular, acetate buffers have been used in experiments designed to determine the rates of carboxymethylation of RNase by bromoacetate (Heinrikson et al, 1965) or iodoacetamide (Fruchter & Crestfield, 1967), the pH dependence of the carboxymethylation reaction (Goren & Barnard, 1970), and the pK values of the histidines at the active site of the enzyme (Meadows et al., 1968;Markley, 1975;Markley & Finkenstadt, 1975;Cohen & Shindo, 1975;Shindo et al, 1976). Chloride ions bind to the histidines at the active site of RNase (Saroff & Carroll, 1962), and acetate probably binds to the active site of the enzyme and inhibits alkylation of RNase by haloacetates.…”

“…This apparent discrepancy implies that the assumption that the ratio of alkylated products formed is simply a reflection of the relative amounts of unprotonated histidines-12 and -119 is incorrect. If one uses, however, the microscopic pK values of Markley & Finkenstadt (1975), it is possible to calculate a value of 0.537 for the ratio of enzyme with unprotonated histidine-1 19 to enzyme with unprotonated histidine-12, where the opposite histidine is protonated. Since the ratio of carboxymethylated histidine-11 9 to carboxymethylated histidine-12 is 4.4, it appears that unprotonated His-1 19 is about 8.3 times more reactive than unprotonated His-12.…”

Proposed rigidity of staphylococcal protease. Comments

“…It is proposed that the unusual low-field resonance observed in proton magnetic resonance spectra of staphylococcal protease (Markley, J. L., Finkenstadt, W. R., Dugas, H., Leduc, P., Drapeau, G. R. (1975), Biochem-In a recent publication (Markley et at, 1975) an unusual resonance was observed in the proton magnetic resonance ( NMR) spectrum of staphylococcal protease. This resonance (labeled X4), at ca.…”

Proton magnetic resonance study of the histidines in hemerythrin and chemical identification of the nonligand histidines

“…It is unlikely that H,, H2, and H3 are incompletely exchanged amide protons because amide protons would not titrate in the pH range in which these protons titrate (Markley et al, 1975).…”