Theories of enzyme specificity and their application to proteases and aminoacyl-Transfer RNA synthetases

Bosshard, Hans Rudolf

doi:10.1007/bf01933911

Cited by 12 publications

(3 citation statements)

References 75 publications

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“…These enzymes display an extremely high degree of specificity as shown by the low frequency of errors found in protein sequences (47). The term 'superspecificity' has been applied to these enzymes when compared to other enzymes of the metabolism-like proteases (48). This high degree of specificity is achieved by the combination of the kinetic and binding specificity (49).…”

Section: Expanding Trna Recognition Is a Crucial Step For New Aminoac...mentioning

confidence: 99%

Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo

Martin

Barends

Eriani

2004

Nucleic Acids Research

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Aminoacyl-tRNA synthetases (aaRSs) are enzymes that are highly specific for their tRNA substrates. Here, we describe the expansion of a class IIb aaRS-tRNA specificity by a genetic selection that involves the use of a modified tRNA displaying an amber anticodon and the argE(amber) and lacZ(amber) reporters. The study was performed on Escherichia coli aspartyl-tRNA synthetase (AspRS) and amber tRNA(Asp). Nine AspRS mutants able to charge the amber tRNA(Asp) and to suppress the reporter genes were selected from a randomly mutated library. All the mutants exhibited a new amber tRNA(Asp) specificity in addition to the initial native tRNA(Asp). Six mutations were found in the anticodon-binding site located in the N-terminal OB-fold. The strongest suppressor was a mutation of residue Glu-93 that contacts specifically the anticodon nucleotide 34 in the crystal structure. The other mutations in the OB-fold were found at close distance from the anticodon in the so-called loop L45 and strand S1. They concern residues that do not contact tRNA(Asp) in the native complex. In addition, this study shows that suppressors can carry mutations located far from the anticodon-binding site. One such mutation was found in the synthetase hinge-module where it increases the tRNA(Asp)-charging rate, and two other mutations were found in the prokaryotic-specific insertion domain and the catalytic core. These mutants seem to act by indirect effects on the tRNA acceptor stem binding and on the conformation of the active site of the enzyme. Altogether, these data suggest the existence of various ways for modifying the mechanism of tRNA discrimination.

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Section: Expanding Trna Recognition Is a Crucial Step For New Aminoac...mentioning

confidence: 99%

Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo

Martin

Barends

Eriani

2004

Nucleic Acids Research

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“…Enzyme-RNA association rate constants measured with enzymatic assays usually report functional interactions, that is, interactions that lead to an enzymatic step. Enzymes can also engage in non-productive binding, where RNA associates with the enzyme, but no enzymatic step occurs (Bosshard, 1976). Non-productive events can be biologically significant, and can ensure substrate specificity (McClain, 1993).…”

Section: Enzymatic Approaches To Measure the Kinetics Of Rna-protein Interactionsmentioning

confidence: 99%

Approaches for measuring the dynamics of RNA–protein interactions

Licatalosi

Jankowsky

2019

WIREs RNA

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RNA-protein interactions are pivotal for the regulation of gene expression from bacteria to human. RNA-protein interactions are dynamic; they change over biologically relevant timescales. Understanding the regulation of gene expression at the RNA level therefore requires knowledge of the dynamics of RNA-protein interactions. Here, we discuss the main experimental approaches to measure dynamic aspects of RNA-protein interactions. We cover techniques that assess dynamics of cellular RNA-protein interactions that accompany biological processes over timescales of hours or longer and techniques measuring the kinetic dynamics of RNA-protein interactions in vitro.

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“…Specificity for a particular substrate is often defined as its catalytic efficiency or specificity constant (k cat /K m ) [38]. Selectivity is expressed as the catalytic efficiency of one substrate to another by the ratio k cat /K m of two substrates [39][40][41] or as the difference in catalytic proficiency which is the ratio of k cat /K m to nonenzymatic acetylation rate, k nE [42,43]. Both k cat /K m and k nE are second order rate constants (mM 21 min 21 ) and thus both catalytic proficiency ((k cat /K m )/k nE ) and the ratio of two specificity constants ((k cat /K m ) A /(k cat /K m ) B ) have no units.…”

Section: Quantitating Specificity and Selectivity Of Multiple Acetyla...mentioning

confidence: 99%

Correction: Quantitating the Specificity and Selectivity of Gcn5-Mediated Acetylation of Histone H3

Kuo¹,

Andrews²

2013

PLoS ONE

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Lysine acetyltransferases (KATs) play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14.K9 < K23. K18. K27 < K36) and the catalytic efficiency (k cat /K m ) for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to 24 kcal/mol between K14 and K18, the highest and lowest measurable k cat /K m . These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3) by Gcn5.

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Theories of enzyme specificity and their application to proteases and aminoacyl-Transfer RNA synthetases

Cited by 12 publications

References 75 publications

Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo

Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo

Approaches for measuring the dynamics of RNA–protein interactions

Correction: Quantitating the Specificity and Selectivity of Gcn5-Mediated Acetylation of Histone H3

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