Stereochemistry of ATP and GTP bound to fish haemoglobins: A transferred nuclear overhauser enhancement, 31P-nuclear magnetic resonance, oxygen equilibrium and molecular modelling study

Abstract

This study was undertaken in order to test the models of ATP and GTP binding to carp deoxyhaemoglobin proposed by Perutz & Brunori (1982) and to find out why GTP is a more potent allosteric effector than ATP. We have determined the conformations of both nucleoside triphosphates by nuclear magnetic resonance studies and found them to be the same. The purines are in anti conformation about the glycosidic bond that links them to the ribose; the pentose ring is 3′- endo; the PO5′C5′C4′ torsion angle lies in the trans domain (180 ° ± 20 °); the P α OP β and P βOP γ angles are as in the free nucleotides, i.e. the trinucleotide chain is fully extended. Models having this conformation were fitted, first manually and then by energy refinement, to the effector site of an atomic model of human deoxyhaemoglobin in which the side-chains in the NA, EF and H segments had been replaced by those of carp. The results showed the location of the polar groups in carp haemoglobin to be such that (PO 4) γ can accept hydrogen bonds from Val NA1 β 2 and from Arg H21β1, while (PO 4) β and (PO 4) α can accept hydrogen bonds from Lys EF6 β 1 and β 2. In ATP, the 6-amino group of the purine can donate a hydrogen bond to Glu NA2 β 1. In GTP, the 2-amino group can donate a hydrogen bond to Glu NA2 β 1; in addition, Val Na1 β 1 can donate a hydrogen bond to O2′ of the ribose. This additional hydrogen bond may explain why in carp haemoglobin GTP is a stronger allosteric effector than ATP. We have found the influence of the two allosteric effectors on the oxygen affinity of trout IV haemoglobin to be the same, even though the only difference in the lining of the allosteric effector sites lies in the replacement of Glu Na2β in carp by Asp in trout IV haemoglobin. Model building then showed that formation of a hydrogen bond between Asp Na2β and the 2-amino group of guanine precludes formation of a hydrogen bond between Val NA1β and O2′ of the ribose or vice versa, which makes the number of hydrogen bonds formed between trout IV haemoglobin and GTP the same as those formed with ATP.