Spectrophotometric titration of tyrosyl residues in alkaline mesentericopeptidase.

At pH 7.0 the alkaline mesentericopeptidase has ultraviolet absorption spectrum with a minimum at 251 nm and a maximum at 280 nm and no visible absorption. From the tyrosine to tryptophan ratio a value of 3 tryptophyl residues per mole of protein is obtained. The molar extinction coefficient at 280 nm is 3.55 X 10(4)M-1cm-1. Spectrophotometric titration studies show that the molecule of mesentericopeptidase contains seven phenolic groups with a pKapp - 9.92 and four to five groups with a pKapp = 11.96. Denaturing agents, such as 5 M guanidine hydrochloride or alkali, normalize the ionization of the tyrosyl residues. There is a good correlation between the spectrophotometric titration data and the results for the reactivities of the tyrosines in mesentericopeptidase towards tetranitromethane. The correlation is explained by the mechanism of nitration. Conclusions about the state of the tyrosyl residues and the three-dimensional structure of mesentericopeptidase are made.     

Essential tyrosyl residues of human placental alkaline phosphatase

• 1.1. Human placental alkaline phosphatase was inactivated with tetranitromethane in a biphasic process.
• 2.2. Spectral and amino acid analysis demonstrated that the inactivation was due to the conversion of tyrosine residues to 3-nitrotyrosine.
• 3.3. The inactivation process showed saturation kinetics.
• 4.4. Protection of the enzyme against tetranitromethane inactivation was afforded by inorganic phosphate.
• 5.5. The binding affinity between the modified enzyme and inorganic phosphate was decreased.
• 6.6. Our results suggest the involvement of tyrosyl residues in the locus of phosphoryl site of the phosphorylated enzyme forms.

     

The ATP-binding site of the human placental H+ pump contains essential tyrosyl residues

Transient exposure of human placental brush-border membrane vesicles to cholate reorients the ATP-driven H+ pump, enabling the pump to transport H+ into the vesicles upon addition of ATP to the external medium. H+ uptake can be measured in these vesicles by following the decrease in the absorbance of acridine orange, a delta pH indicator. We investigated the role of tyrosyl residues in the catalytic function of the H+ pump by studying the effects of tyrosyl group specific reagents on ATP-driven H+ uptake in cholate-pretreated membrane vesicles. The reagents tested were 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), N-acetylimidazole, tetranitromethane, and p-nitrobenzenesulfonyl fluoride. Treatment of the membrane vesicles with these reagents resulted in the inhibition of the ATP-driven H+ uptake, and the inhibitory potency was in the following order: NBD-Cl greater than tetranitromethane greater than p-nitrobenzenesulfonyl fluoride greater than N-acetylimidazole. The inhibition of the H+ pump by NBD-Cl was reversible by 2-mercaptoethanol, and the inhibition by N-acetylimidazole was reversible by hydroxylamine. Since these reagents are not absolutely specific for tyrosyl groups and can also react with thiol groups, we studied the interaction of N-acetylimidazole with the H+ pump whose triol groups were masked by reaction with p-(chloromercuri)benzenesulfonate. The SH-masked pump was totally inactive, but the activity could be restored by dithiothreitol. On the contrary, the activity of the SH-masked H+ pump which was subsequently treated with N-acetylimidazole could not be restored by dithiothreitol, suggesting that thiol groups were not involved in the inhibition of the H+ pump by N-acetylimidazole.(ABSTRACT TRUNCATED AT 250 WORDS)     

The status of tyrosyl residues in a Formosan cobra cardiotoxin.

Spectrophotometric titration of Formosan cobra cardiotoxin showed that two of the three tyrosyl residues were titrated freely with a normal apparent pKa of 9.6 whereas the remaining one ionized at pH above 11.0. Nitration of cardiotoxin in Tris . HCl buffer with tetranitromethane resulted in the selective nitration of tyrosine 11 and tyrosine 22. It also revealed that tyrosine 51 was the abnormal one in the spectrophotometric titration. Complete nitration occurred in the presence of 6.0 M guanidine hydrochloride. Compared with the conformation of native cardiotoxin, the peptide conformation of the partially nitrated cardiotoxin did not change significantly but the conformation of the completely nitrated cardiotoxin changed remarkably. The biological activity of cardiotoxin was indeed affected by nitration, but the immunological activity was nearly intact even when all the tyrosine residues were nitrated.     

The tyrosyl residues in creatine kinase. Modification by iodine.

The effect of the iodination of tyrosyl residues in creatine kinase from rabbit muscle has been investigated at alkaline pH after reversible masking of the reactive thiol groups. The conversion of 4-5 tyrosyl residues to monoiodotyrosines as measured by spectrotitration and by radioactive iodine labelling resulted in almost total loss of enzymic activity. The modified enzyme was unable to bind its nucleotide substrates but no significant conformational change was revealed by optical rotatory dispersion or Stokes radius measurements. However, change in the reactivity of some non-essential thiol groups, presumably those located near the active thiol groups, was observed.     

Essential tyrosyl residues in Lactobacillus casei thymidylate synthetase

Sulfhydryl-blocked thymidylate synthetase (EC 2.1.1.4.5) is rapidly inactivated by low concentrations of tetranitromethane. This reagent first nitrates two non-essential tyrosines per dimeric enzyme molecule followeed by two essential tyrosines with no oxidation of sulfhydryl groups. dUMP affords significant protection against inactivation. These results suggest that essential tyrosyl residues are present in the active sites of the enzyme.     

pH-jump titration of the tyrosyl groups of bovine plasma albumin

Ionization properties of the tyrosyl groups of bovine plasma albumin in various conformational states—the native state (N), the two acid states (F and E), and the state (B) stable at slightly alkaline pH—were studied by means of a stopped-flow-pH-jump technique. The technique allows us to obtain the tyrosyl titration curve in a conformational state that is unstable in the pH region of the titration. The pH jumps from the N and B states to various alkaline pH's, where the tyrosines ionize to bring about a time-dependent increase in absorption at 296 nm, indicating that a number of the tyrosines buried initially become susceptible to ionization as a result of the alkaline transition occurring above pH 10.8. Extrapolation of the observed absorption change to zero time gives a spectrophotometric titration curve in the initial conformational state. Only 30–401% of the 19 tyrosines of the protein can ionize both in the N and the B state at pH 12. The pH jumps from the F and E states, on the other hand, give a decrease in absorption between pH 9 and 11.7, indicating that the tyrosyl groups initially exposed are remarked by refolding after the pH jumps, and the zero-time titration curves show that essentially all the tyrosyl groups ionize normally in these acid states. The results are discussed in relation to the known results of the tyrosyl exposure of the protein measured by other techniques, and the consistency among them demonstrates the effectiveness of the pH-jump titration method. Hydrogen bonding between the abnormal tyrosyl and carboxylate groups as a mechanism to stabilize native albumin is suggested from characteristics of the alkaline transition, which also involves the exposure of the tyrosyl groups, and from the tyrosyl titration curves in the native and acid states.