Role of hydrophobicity of phenylalanine beta 85 and leucine beta 88 in the acceptor pocket for valine beta 6 during hemoglobin S polymerization.
Characterization of the hydrophobic EF acceptor pocket involving Phe-beta 85 and Leu-beta 88 as well as the Val-beta 6 donor site is critical for understanding the polymerization of deoxy Hb S. Glu substitutions at beta 85 or beta 88 in Hb S were made and expressed in yeast in an effort to evaluate the role of hydrophobicity in the acceptor pocket during polymerization of Hb S. Both substitutions result in decreased tetramer stability, increases in oxygen affinity, and inhibition in polymerization compared with Hb S. Critical concentrations for polymerization of Hb SF beta 85E and Hb SL beta 88E were 2.4- and 7-fold higher, respectively, than that of Hb S, while the value for Hb SL beta 88E was intermediate between those previously reported for Hb SL beta 88A and Hb SL beta 88F (Adachi, K., Konitzer, P., Paulraj, C. G., and Surrey, S. (1994) J. Biol. Chem. 269, 17477-17480). Kinetics of polymerization of Glu-beta 85 and Glu-beta 88 deoxy Hb S tetramers were biphasic at lower hemoglobin concentrations like deoxy Hb SL beta 88A, suggesting formation of two types of polymers during polymerization. The time required to form half the total amount of polymer (t1/2) for deoxy Hb SF beta 85E was 10-fold shorter than that for deoxy Hb SL beta 88E. In addition, t1/2 for deoxy Hb SF beta 85E was 2.5-fold shorter, while that for Hb SL beta 88E was 4-fold longer than deoxy Hb SL beta 88A at equivalent concentrations. These results suggest that hydrophobicity of the amino acid at beta 88 appears more critical than that at beta 85 in the acceptor pocket for Val-beta 6. Furthermore, stereospecificity of the acceptor pocket in addition to hydrophobicity of beta 88 are critical for stable hydrophobic interactions with Val-beta 6 during deoxy Hb S polymerization.
Adachi
K
K
Division of Hematology, University of Pennsylvania School of Medicine, Children's Hospital of Philadelphia, Pennsylvania 19104.
Reddy
L R
LR
Surrey
S
S
D005914:Globins / Q000737:chemistry*; D006451:Hemoglobin, Sickle / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D066298:In Vitro Techniques; D007930:Leucine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011108:Polymers; D011485:Protein Binding; D011489:Protein Denaturation; D012995:Solubility; D013329:Structure-Activity Relationship; D014633:Valine / Q000737:chemistry; D014867:Water
D006451:Hemoglobin, Sickle; D011108:Polymers; D014867:Water; D010649:Phenylalanine; D005914:Globins; D007930:Leucine; D014633:Valine