Mechanism of adenylate kinase. Demonstration of a functional relationship between aspartate 93 and Mg sup 2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR

Journal Article · Tue Jun 04 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00236a029· OSTI ID:5026200

Yan, Honggao; Tsai, Mingdaw ^[1]

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1. Ohio State Univ., Columbus (United States)

Earlier magnetic resonance studies suggested no direct interaction between Mg{sup 2+} ions and adenylate kinase (AK) in the AK{center dot}MgATP (adenosine 5'-triphosphate) complex. However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg{sup 2+} ion in the muscle AK {center dot} MgATP complex. On the other hand, in the 2.6-{angstrom} crystal structure of the yeast AK{center dot}MgAP{sub 5}A (P{sup 1}, P{sup 5}-bis(5'-adenosyl)pentaphosphate) complex, the Mg{sup 2+} ion is in proximity to aspartate 93. Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in K{sub m}, and a 650-fold decrease in k{sub cat}. Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues. However, the results of detailed analyses of the free enzymes and the AK{center dot}MgAP{sub 5}A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations. Thus it is concluded that Asp-93 stabilizes the transition state by ca. 3.9 kcal/mol. Other results raised the question of whether Mg{sup 2+} could bind to D93A{center dot}nucleotide complexes, which was then probed by {sup 25}MgNMR. The results suggest that Mg{sup 2+} does bind to the D93A{center dot}AP{sub 5}A complex, but possibly only weakly.

• OSTI ID: 5026200
• Journal Information: Biochemistry; (United States), Vol. 30:22; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ASPARTIC ACID
NUCLEAR MAGNETIC RESONANCE
PHOSPHOTRANSFERASES
BIOCHEMICAL REACTION KINETICS
CHEMICAL SHIFT
HISTIDINE
MAGNESIUM 25
MAGNESIUM COMPOUNDS
MUTAGENESIS
OVERHAUSER EFFECT
PHOSPHORUS 31
ALKALINE EARTH ISOTOPES
ALKALINE EARTH METAL COMPOUNDS
AMINO ACIDS
AZOLES
CARBOXYLIC ACIDS
ENZYMES
EVEN-ODD NUCLEI
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
IMIDAZOLES
ISOTOPES
KINETICS
LIGHT NUCLEI
MAGNESIUM ISOTOPES
MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
REACTION KINETICS
RESONANCE
STABLE ISOTOPES
TRANSFERASES
550201* - Biochemistry- Tracer Techniques