Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine

Journal Article · Tue Jul 10 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00479a007· OSTI ID:6037975

Yan, Honggao; Shi, Zhengtao; Tsai, Mingdaw ^[1]

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1. Ohio State Univ., Columbus (USA)

Replacement of the arginine-138 of adenylate kinase (AK) by lysine or methionine resulted in a decrease in k{sub cat} by a factor of 10{sup 4}, increases in K{sub m} by a factor of 10-20, and relatively little changes in dissociation constants. Proton nuclear magnetic resonance (NMR) studies were then undertaken to obtain structural information for quantitative interpretation of the kinetic data. Since the lysine mutant (R138K) represents a conservative mutation with surprisingly large effects on kinetics, structural studies were focused on the wild type (WT) and R138K. The results and conclusions are summarized as follows: (i) The aromatic spin systems of WT and R138K were assigned from total correlated spectroscopy (TOCSY). (ii) Proton NMR titrations with AMP and MgATP suggested that substrate binding affinities and substrate-induced conformational changes are nearly identical between WT and R138K. (iii) Notable differences were observed between the proton NMR spectra of the WT and R138K complexes with the reaction mixture, which agrees with the perturbation in the K{sub m} values of R138K. (iv) Qualitative comparison of the NOESY cross peaks between aliphatic side chains and aromatic protons indicates that the patterns are almost identical between free WT and free R138K. (v) The above kinetic and structural results led to the conclusion that Arg-138 stabilizes the ternary complexes by 1.4-1.8 kcal/mol and stabilizes the transition state by at least 7 kcal/mol and that the critical functional role of Arg-138 cannot be replaced by lysine. (vi) Since Arg-138 is distant from the substrate sites proposed from previous NMR studies serious revision will be required for this model.

• OSTI ID: 6037975
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:27; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ADENYLIC ACID
AMINO ACIDS
ARGININE
BACTERIA
BARYONS
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CATALYSIS
DRUGS
ELEMENTARY PARTICLES
ENZYMES
ESCHERICHIA COLI
FERMIONS
HADRONS
KINETICS
LIPOTROPIC FACTORS
LYSINE
MAGNETIC RESONANCE
METHIONINE
MICROORGANISMS
MUTANTS
NUCLEAR MAGNETIC RESONANCE
NUCLEONS
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PROTONS
PURIFICATION
REACTION KINETICS
RESONANCE
TRANSFERASES