Proton Nuclear Magnetic Resonance Studies of Ribonuclease A in H 2 O

Aromatic amino acid residues in epidermal growth factor (EGF) isolated from the rat have been investigated by proton n.m.r. and nuclear Overhauser methods at 500 MHz and by photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) experiments at 360 MHz. Rat EGF contains six aromatic residues, i.e. one histidine and five tyrosine residues. pH titration data allow identification of the histidine imidazole ring protons, whereas two-dimensional n.m.r. correlated spectroscopy establishes connectivities between tyrosine ring (2,6) and (3,5) proton resonances. Photo-c.i.d.n.p. data give evidence for solvent exposure of the one histidine and the five tyrosine residues in rat EGF. Nuclear Overhauser experiments and pH titration data suggest proximity relationships among four of the tyrosine residues and the histidine residue. These data indicate the presence of a clustered, aromatic, structural domain on the protein surface and may provide a clue to the understanding of the functional structure of EGF.

Section: Resultsmentioning

confidence: 99%

Structural characterization and exposure of aromatic residues in epidermal growth factor from the rat

Mayo¹,

Schaudies²,

Savage³

et al. 1986

“…Exchangeable protons that are not seen when 2H20 is used as solvent may be seen when 1H20 is used, provided that the residence time of the proton on a nitrogen atom is of the order of milliseconds (or longer). Protons of OH and SH groups usually exchange too rapidly to be seen (Glickson et al, 1971) and freely exchanging protons of imidazole NH group are also in rapid exchange with the solvent and will not be seen (Patel et al, 1972;Griffin et al, 1973). Peptide NH groups usually give resonances at about 7.7-9.7p.p.m., the tryptophan NH group at 9.7-10.7p.p.m.…”

Section: -Phosphoglycollatementioning

confidence: 99%

Enzyme-substrate and enzyme-inhibitor complexes of triose phosphate isomerase studied by 31P nuclear magnetic resonance

Campbell¹,

Jones²,

Kiener³

et al. 1979

The complex formed between the enzyme triose phosphate isomerase (EC 5.3.1.1.), from rabbit and chicken muscle, and its substrate dihydroxyacetone phosphate was studied by 31P n.m.r. Two other enzyme-ligant complexes examined were those formed by glycerol 3-phosphate (a substrate analogue) and by 2-phosphoglycollate (potential transition-state analogue). Separate resonances were observed in the 31P n.m.r. spectrum for free and bound 2-phosphoglycollate, and this sets an upper limit to the rate constant for dissociation of the enzyme-inhibitor complex; the linewidth of the resonance assigned to the bound inhibitor provided further kinetic information. The position of this resonance did not vary with pH but remained close to that of the fully ionized form of the free 2-phosphoglycollate. It is the fully ionized form of this ligand that binds to the enzyme. The proton uptake that accompanies binding shows protonation of a group on the enzyme. On the basis of chemical and crystallographic information [Hartman (1971) Biochemistry 10, 146--154; Miller & Waley (1971) Biochem. J. 123, 163--170; De la Mare, Coulson, Knowles, Priddle & Offord )1972) Biochem. J. 129, 321--331; Phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc. Trans. 5, 642--647] this group is believed to be glutamate-165. On the other hand, the position of the resonance of D-glycerol 3 phosphate (sn-glycerol 1-phosphate) in the enzyme-ligand complex changes with pH, and both monoanion and dianon of the ligand bind, although dianion binds better. The substrate, dihydroxyacetone phosphate, behaves essentially like glycerol 3-phosphate. The experiments with dihydroxy-acetone phosphate and triose phosphate isomerase have to be carried out at 1 degree C because at 37 degrees C there is conversion into methyl glyoxal and orthophosphate. The mechanismof the enzymic reaction and the reasons for rate-enhancement are considered, and aspects of the pH-dependence are discussed in an Appendix.

“…(2) Thus 1/r does not fall monotonically as the pH is raised, but instead has a minimum of 10s-' at pH7. The relaxation time for protonation of a histidine residue in ribonuclease has been measured directly in unbuffered solution (Patel et al, 1972), and is indeed of the order of 0.1 ms.…”

Section: Theory Relaxation Time For Ionizationmentioning

confidence: 99%

Estimation of the dissociation constants of enzyme-substrate complexes from steady-state measurements. Interpretation of pH-independence of Km

Cornish‐Bowden

1976

If the Michaelis constant of an enzyme-catalysed reaction is independent of pH under conditions where the catalytic constant varies with pH, it is equal to the thermodynamic dissociation constant of the enzyme-substrate complex. This is true for realistic mechanisms in which binding and catalytic steps, are clearly distinguished, as well as for the simpler mechanisms that have been considered previously. It is also true for a mechanism in which a bell-shaped pH profile for the catalytic constant results from a change of rate-limiting step with pH. The relaxation time for ionization of a typical group in unbuffered solutions at 25 degrees C is of the order of 0.1 ms at the longest, and is much shorter in buffered solutions. Thus ionizations in almost all enzyme mechanisms can properly be treated as equilibria, provided that ionization is not accompanied by a slow, compulsory change in conformation.