Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues

Zaza A. Khuchua, Wenning Qin, Jaime Boero, Judy Cheng, R. Payne, Valdur A. Saks, Arnold W. Strauss

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.

Original languageEnglish (US)
Pages (from-to)22990-22996
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number36
DOIs
StatePublished - Sep 4 1998
Externally publishedYes

Fingerprint

Mitochondrial Form Creatine Kinase
Mitochondrial Membranes
Mitochondria
Complementary DNA
Membranes
Amino Acids
Neutral Amino Acids
Peptides
Mutation
Creatine
Oxidative Phosphorylation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues. / Khuchua, Zaza A.; Qin, Wenning; Boero, Jaime; Cheng, Judy; Payne, R.; Saks, Valdur A.; Strauss, Arnold W.

In: Journal of Biological Chemistry, Vol. 273, No. 36, 04.09.1998, p. 22990-22996.

Research output: Contribution to journalArticle

Khuchua, Zaza A. ; Qin, Wenning ; Boero, Jaime ; Cheng, Judy ; Payne, R. ; Saks, Valdur A. ; Strauss, Arnold W. / Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 36. pp. 22990-22996.
@article{5a79640a009746e4ba9ee495e0a3d35e,
title = "Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues",
abstract = "Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.",
author = "Khuchua, {Zaza A.} and Wenning Qin and Jaime Boero and Judy Cheng and R. Payne and Saks, {Valdur A.} and Strauss, {Arnold W.}",
year = "1998",
month = "9",
day = "4",
doi = "10.1074/jbc.273.36.22990",
language = "English (US)",
volume = "273",
pages = "22990--22996",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "36",

}

TY - JOUR

T1 - Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues

AU - Khuchua, Zaza A.

AU - Qin, Wenning

AU - Boero, Jaime

AU - Cheng, Judy

AU - Payne, R.

AU - Saks, Valdur A.

AU - Strauss, Arnold W.

PY - 1998/9/4

Y1 - 1998/9/4

N2 - Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.

AB - Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.

UR - http://www.scopus.com/inward/record.url?scp=0032483521&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032483521&partnerID=8YFLogxK

U2 - 10.1074/jbc.273.36.22990

DO - 10.1074/jbc.273.36.22990

M3 - Article

C2 - 9722522

AN - SCOPUS:0032483521

VL - 273

SP - 22990

EP - 22996

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 36

ER -