A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia.
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 Km and Vmax for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 microM and 18.5 mumol/(min.mg), respectively. Deletion of Leu at the -5 position lowers the Vmax/Km by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in Vmax/Km 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 Vmax/Km. In contrast, substitution with Leu increases Vmax/Km by 1.8-fold. Arg at -3 is also required for recognition as shown by an approximately 240-fold decrease in Vmax/Km 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 Vmax/Km by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase Vmax/Km 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.
Lee
J C
JC
Department of Pharmacology and Therapeutics, State University of New York at Buffalo 14214.
Kwon
Y G
YG
Lawrence
D S
DS
Edelman
A M
AM
D000595:Amino Acid Sequence; D000599:Amino Acids, Diamino; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D051381:Rats; D012694:Serine; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine; D016704:Synapsins / Q000737:chemistry / Q000378:metabolism*
D000599:Amino Acids, Diamino; D009842:Oligopeptides; D016704:Synapsins; D012694:Serine; D017871:Calcium-Calmodulin-Dependent Protein Kinases