Autoregulation of enzymes by pseudosubstrate prototopes: myosin light chain kinase.
The myosin light chain kinase requires calmodulin for activation. Tryptic cleavage of the enzyme generates an inactive 64-kilodalton (kD) fragment that can be further cleaved to form a constitutively active, calmodulin-independent, 61-kD fragment. Microsequencing and amino acid analysis of purified peptides after proteolysis of the 61- and 64-kD fragments were used to determine the amino-terminal and carboxyl-terminal sequences of the 64-kD fragment. Cleavage within the calmodulin-binding region at Arg505 generates the catalytically inactive 64-kD fragment, which is incapable of binding calmodulin. Further digestion removes a carboxyl-terminal fragment, including the pseudosubstrate sequence Ser484-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met- Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg505 and results in a calmodulin-independent 61-kD fragment. Both the 61- and 64-kD fragments have the same primary amino-terminal sequences. These results provide direct support for the concept that the pseudosubstrate structure binds the active site and that the role of calmodulin is to modulate this interaction. Pseudosubstrates may be utilized in analogous ways by other allosterically regulated enzymes.
Pearson
R B
RB
Department of Medicine, University of Melbourne, Repatriation General Hospital, Heidelberg, Victoria, Australia.
Wettenhall
R E
RE
Means
A R
AR
Hartshorne
D J
DJ
Kemp
B E
BE
D000595:Amino Acid Sequence; D002147:Calmodulin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000032:analysis / Q000378:metabolism*; D010449:Peptide Mapping; D013379:Substrate Specificity
D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase