The Negative Dominant Effects of T340M Mutation on Mammalian Pyruvate Kinase

Abstract

A fundamental issue in allosteric regulatory enzymes is the identification of pathways of signal transmission. Rabbit muscle and kidney pyruvate kinase isozymes are ideal to address this issue because these isozymes exhibit different enzymatic regulatory patterns, and the sequence differences between these isozymes have identified the amino acid residues that alter their kinetic behavior. In an earlier study, Cheng et al. (Cheng, X., Friesen, R. H. E., and Lee, J. C. (1996) J. Biol. Chem. 271, 6313-6321), reported the effects of a threonine to methionine mutation at residue 340 in the muscle isozyme. In this study, the same mutation was effected in the kidney isozyme. Qualitatively, the same negative effects are observed in both isozymes, namely a significant decrease in catalytic efficiency and decrease in apparent affinity for phosphoenolpyruvate but no change in affinity for ADP, and a decrease in responsiveness to the presence of effectors, be it activator or inhibitor. Because the diversity in the primary sequence between these two isozymes does not alter the negative impact of the T340M mutation, it can be concluded that this mutation exerts a dominant, negative effect. The negative effects of T340M mutation on the kinetic properties imply that there is communication between residue 340 and the active site. Residue 340 is located at the 1,4 subunit interface; however, a T340M mutation enhances the dimerization affinity along the 1,2 subunit interface. Thus, this study has identified a communication network among the active site, residue 340, and the 1,2 subunit interface.Mammalian pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) (PK) 1 is a key regulatory glycolytic enzyme which catalyzes the transphosphorylation from phosphoenolpyruvate (PEP) to ADP. One of the enzymatic products, pyruvate, is situated at a major metabolic junction between the metabolic pathways of carbohydrates, amino acids, and lipids. Thus, a tight regulatory control of PK is critical to proper cellular function.PK activity in mammalian cells is regulated by two different mechanisms. The first mechanism occurs at the level of expression. The mammalian genome contains two distinct genes coding for four different enzymes with PK activity. The M 1 -and M 2 -type isozymes of PK are produced from one gene by alternative RNA splicing (1). The R-and L-type PK isozymes are products of a different gene with two different promoters. The R-and L-type mRNAs differ only in their 5Ј-terminal sequences (2). These four isozymic forms of PK are expressed in a tissuespecific manner (3). The M 1 -type PK is the major isozyme of cardiac muscle and brain and is the only isozyme found in adult skeletal muscle. The M 2 -type PK is widely distributed throughout the body and is the major isozyme derived from kidney and leukocytes. The L-type PK is the major isozyme in the liver, and the R-type isozyme is isolated from erythrocytes.The second mechanism of control of PK activity is through allosteric regulation. PK isozymes are regulated by a...

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

“…Thus, these structural data show a communication between these two different subunit interfaces. A similar conclusion was derived from the results of our study of subunit assembly [26,27]. It is evident that a mutation at residue 402 leads to increased dynamics in distant sites through long range communication without significant changes in secondary structures.…”

“…The resultant is a differential effect on the functional energetics of PK. This study demonstrates the linkage between distant residues in different interfacial interactions i.e., establishing the functional coupling among residues and the identities of these residues [24,26,27]. …”

Structure and Function of Human Erythrocyte Pyruvate Kinase

“…Thus, the altered kinetics displayed by the T384M mutant is difficult to rationalize. Modified enzymatic parameters were observed also for the equivalent mutation in the rabbit kidney isozyme (30). Thr 384 is close to the contact region between the A and B domains (Fig.…”

Isotype‐specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation

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