A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia

Cho Lee Jungsook Cho Lee, Y. G. Kwon, D. S. Lawrence, A. M. Edelman

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Abstract

The substrate recognition determinants of Ca2+/calmodulin-dependent protein kinase Ia were investigated by using peptide analogues based on the amino acid sequence around Ser-9 of synapsin I. The K(m) and V(max) for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 μM and 18.5 μmol/(min · mg), respectively. Deletion of Leu at the -5 position lowers the V(max)/K(m) by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in V(max)/K(m) produced by Arg, Ala, or Asp substitutions, but only 2.6-fold decrease after Phe substitution at this position. A hydrophobic residue is similarly required at the +4 position. Deletion of Phe at this position produces a 67-fold reduction, and substitution of Ala for Phe a 43-fold reduction in V(max)/K(m). In contrast, substitution with Leu increases V(max)/K(m) by 1.8- fold. Arg at -3 is also required for recognition as shown by an ≃240-fold decrease in V(max)/K(m) after Ala substitution at this position. Positions - 2, -4, and +1 appear to play secondary roles in substrate recognition. Arg at -2 and -4 are positive determinants, since Ala substitution at these positions decreases V(max)/K(m) by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase V(max)/K(m) by 2.3- and 6.3-fold, respectively. Substitution of Ala for Leu at -1 or Thr for Ser at the 0 position has little effect on phosphorylation kinetics. Thus, Ca2+/calmodulin-dependent protein kinase Ia has the minimal substrate recognition motif of Hyd-Xaa-Arg-Xaa-Xaa- (Ser*/Thr*)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue.

Original languageEnglish
Pages (from-to)6413-6417
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number14
DOIs
Publication statusPublished - 1994 Jul 12

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Basic Amino Acids
Calcium-Calmodulin-Dependent Protein Kinases
Viperidae
Synapsins
Peptides
Amino Acid Sequence
Phosphorylation
Amino Acids

All Science Journal Classification (ASJC) codes

  • General

Cite this

@article{74a8b30a34c54a1cbc15191ed54450dd,
title = "A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia",
abstract = "The substrate recognition determinants of Ca2+/calmodulin-dependent protein kinase Ia were investigated by using peptide analogues based on the amino acid sequence around Ser-9 of synapsin I. The K(m) and V(max) for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 μM and 18.5 μmol/(min · mg), respectively. Deletion of Leu at the -5 position lowers the V(max)/K(m) by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in V(max)/K(m) produced by Arg, Ala, or Asp substitutions, but only 2.6-fold decrease after Phe substitution at this position. A hydrophobic residue is similarly required at the +4 position. Deletion of Phe at this position produces a 67-fold reduction, and substitution of Ala for Phe a 43-fold reduction in V(max)/K(m). In contrast, substitution with Leu increases V(max)/K(m) by 1.8- fold. Arg at -3 is also required for recognition as shown by an ≃240-fold decrease in V(max)/K(m) after Ala substitution at this position. Positions - 2, -4, and +1 appear to play secondary roles in substrate recognition. Arg at -2 and -4 are positive determinants, since Ala substitution at these positions decreases V(max)/K(m) by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase V(max)/K(m) by 2.3- and 6.3-fold, respectively. Substitution of Ala for Leu at -1 or Thr for Ser at the 0 position has little effect on phosphorylation kinetics. Thus, Ca2+/calmodulin-dependent protein kinase Ia has the minimal substrate recognition motif of Hyd-Xaa-Arg-Xaa-Xaa- (Ser*/Thr*)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue.",
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A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia. / Jungsook Cho Lee, Cho Lee; Kwon, Y. G.; Lawrence, D. S.; Edelman, A. M.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, No. 14, 12.07.1994, p. 6413-6417.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia

AU - Jungsook Cho Lee, Cho Lee

AU - Kwon, Y. G.

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N2 - The substrate recognition determinants of Ca2+/calmodulin-dependent protein kinase Ia were investigated by using peptide analogues based on the amino acid sequence around Ser-9 of synapsin I. The K(m) and V(max) for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 μM and 18.5 μmol/(min · mg), respectively. Deletion of Leu at the -5 position lowers the V(max)/K(m) by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in V(max)/K(m) produced by Arg, Ala, or Asp substitutions, but only 2.6-fold decrease after Phe substitution at this position. A hydrophobic residue is similarly required at the +4 position. Deletion of Phe at this position produces a 67-fold reduction, and substitution of Ala for Phe a 43-fold reduction in V(max)/K(m). In contrast, substitution with Leu increases V(max)/K(m) by 1.8- fold. Arg at -3 is also required for recognition as shown by an ≃240-fold decrease in V(max)/K(m) after Ala substitution at this position. Positions - 2, -4, and +1 appear to play secondary roles in substrate recognition. Arg at -2 and -4 are positive determinants, since Ala substitution at these positions decreases V(max)/K(m) by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase V(max)/K(m) by 2.3- and 6.3-fold, respectively. Substitution of Ala for Leu at -1 or Thr for Ser at the 0 position has little effect on phosphorylation kinetics. Thus, Ca2+/calmodulin-dependent protein kinase Ia has the minimal substrate recognition motif of Hyd-Xaa-Arg-Xaa-Xaa- (Ser*/Thr*)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue.

AB - The substrate recognition determinants of Ca2+/calmodulin-dependent protein kinase Ia were investigated by using peptide analogues based on the amino acid sequence around Ser-9 of synapsin I. The K(m) and V(max) for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 μM and 18.5 μmol/(min · mg), respectively. Deletion of Leu at the -5 position lowers the V(max)/K(m) by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in V(max)/K(m) produced by Arg, Ala, or Asp substitutions, but only 2.6-fold decrease after Phe substitution at this position. A hydrophobic residue is similarly required at the +4 position. Deletion of Phe at this position produces a 67-fold reduction, and substitution of Ala for Phe a 43-fold reduction in V(max)/K(m). In contrast, substitution with Leu increases V(max)/K(m) by 1.8- fold. Arg at -3 is also required for recognition as shown by an ≃240-fold decrease in V(max)/K(m) after Ala substitution at this position. Positions - 2, -4, and +1 appear to play secondary roles in substrate recognition. Arg at -2 and -4 are positive determinants, since Ala substitution at these positions decreases V(max)/K(m) by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase V(max)/K(m) by 2.3- and 6.3-fold, respectively. Substitution of Ala for Leu at -1 or Thr for Ser at the 0 position has little effect on phosphorylation kinetics. Thus, Ca2+/calmodulin-dependent protein kinase Ia has the minimal substrate recognition motif of Hyd-Xaa-Arg-Xaa-Xaa- (Ser*/Thr*)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue.

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