Proton‐Magnetic‐Resonance Studies of Complexes of Pancreatic Ribonuclease A with Pyrimidine and Purine Nucleotides

Abstract

In continuation of earlier work new results on the behaviour of certain proton resonances of pancreatic ribonuclease A and of nucleotides during complex formation are reported. By varying the structure of the base or the position of the phosphate group at the ribose moiety it can be shown that the main structural feature of the ribonuclease · pyrimidine‐nucleotide complexes is maintained. However, the microscopic binding constants of the interactions of the various parts of a nucleotide with the corresponding amino acid sidegroups are different for nearly all the investigated complexes. While the modified purine base of 8‐oxoadenosine 3′‐phosphate seems to be bound in the pyrimidine binding site of ribonuclease A also, the base of the other adenosine nucleotides studied occupy other sites at the enzyme. For adenosine 3′‐ and 5′‐monophosphate this might be the binding site shown to be specific for purine bases by X‐ray crystallography. But the results obtained for the ribonuclease · adenosine‐2′‐phosphate complex suggest that the adenosine nucleotides do not bind exclusively in this purine binding site.

The Reaction of Bovine Pancreatic Ribonuclease A with 6‐Chloropurineriboside 5′‐Monophosphate

“…In the complex of ribonuclease S and S'AMP, B2, R2 and pl are occupied whereas in the complex with 3'AMP, Bz and R2 are occupied but the phosphate seems to be placed in a site other than pl [29]. The different binding of 5'AMP and 3'AMP has also been detected by NMR experiments [7] which support this intepretation.…”

“…At more basic pH values the K, is smaller because of the loss of the protons from the active-centre histidines responsible for the binding of the phosphate group [7,18,19]. For this reason, although at higher pH values the nucleophilic attack is more efficient, as seen from the loss of activity, it is probably less specific.…”

“…A number of studies using several techniques such as difference spectroscopy [4,5], NMR spectroscopy [6,7] and X-ray crystallography [8 -101 have tried to elucidate the nature of this purine-binding site, its existence being now widely accepted. The site appears to be of a partially hydrophobic character [S] and, from the X-ray work, it is likely that Gln-69, Asn-71, Glu-111, His-119 and Asp-121 belong, or are close, to it.…”

15N‐ and 1H‐NMR investigations of the active‐site amino acids in semisynthetic RNase S′ and RNase A

“…The position of the phosphate group in uridine-nucleotide complexes could be similar to that in the S'AMP complex, where the pK value of His-12 is likewise affected only to a slight extent. The high binding constants [I] and the pKvalue of His-1 19 [64) indicate a good binding of base and phosphate moiety. Recently published X-ray structures support this assumption.…”

Infrared studies on ribonuclease a‐mononucleotide complexes ionization states and hydrogen bonding of the phosphate groups

“…The band shifts reflect the lowering of the symmetry of the phosphate groups which can be taken as evidence for the involvement of the phosphate groups of Cyd-2'P in binding to the enzyme. From X-ray and NMR studies it is assumed that His 12* form hydrogen bonds with the phosphate moiety of Cyd-2'-P [2, 3,8]. These hydrogen bonds can be represented by the following equilibrium** * Note the reversal of the 'H-NMR peak assignments of…”