TY - JOUR
T1 - A lead-dependent DNAzyme with a two-step mechanism
AU - Brown, Andrea K.
AU - Li, Jing
AU - Pavot, Caroline M.B.
AU - Lu, Yi
N1 - Copyright:
Copyright 2011 Elsevier B.V., All rights reserved.
PY - 2003/6/17
Y1 - 2003/6/17
N2 - A detailed biochemical and mechanistic study of in vitro selected variants of 8-17 DNAzymes is presented. Even though the 8-17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg2+ (called 8-17), 0.5 mM Mg2+/50 mM histidine (called Mg5), or 100 μM Zn2+ (called 17E), all variants are shown to be the most active with Pb2+ (8-17: kobs ∼0.5 min-1; Mg5: kobs ∼2 min-1; 17E: kobs ∼1 min-1 with 200 μM Pb2+ at pH 5.0). For the 17E variant of the 8-17 DNAzyme, the single-turnover rate constants followed the order of Pb2+ ≫ Zn2+ ≫ Mn2+ ≈ Co2+ > Ni2+ > Mg2+ ≈ Ca2+ > Sr2+ ≈ Ba2+. The catalytic rate is half-maximal at 13.5 μM Pb2+, 0.97 mM Zn2+, or 10.5 mM Mg2+, suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb2+ > Zn2+ > Mg2+. The Pb2+-dependent activity increases linearly with pH and the slope of the plot of log kobs versus pH is ∼1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G•T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg2+ and Zn2+ catalyze only a transesterification reaction with formation of a product containing a 2′,3′-cyclic phosphate, Pb2+ catalyzes a transesterification reaction followed by hydrolysis of the 2′,3′-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8-17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb2+-binding sites and their roles in the two-step mechanism.
AB - A detailed biochemical and mechanistic study of in vitro selected variants of 8-17 DNAzymes is presented. Even though the 8-17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg2+ (called 8-17), 0.5 mM Mg2+/50 mM histidine (called Mg5), or 100 μM Zn2+ (called 17E), all variants are shown to be the most active with Pb2+ (8-17: kobs ∼0.5 min-1; Mg5: kobs ∼2 min-1; 17E: kobs ∼1 min-1 with 200 μM Pb2+ at pH 5.0). For the 17E variant of the 8-17 DNAzyme, the single-turnover rate constants followed the order of Pb2+ ≫ Zn2+ ≫ Mn2+ ≈ Co2+ > Ni2+ > Mg2+ ≈ Ca2+ > Sr2+ ≈ Ba2+. The catalytic rate is half-maximal at 13.5 μM Pb2+, 0.97 mM Zn2+, or 10.5 mM Mg2+, suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb2+ > Zn2+ > Mg2+. The Pb2+-dependent activity increases linearly with pH and the slope of the plot of log kobs versus pH is ∼1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G•T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg2+ and Zn2+ catalyze only a transesterification reaction with formation of a product containing a 2′,3′-cyclic phosphate, Pb2+ catalyzes a transesterification reaction followed by hydrolysis of the 2′,3′-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8-17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb2+-binding sites and their roles in the two-step mechanism.
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U2 - 10.1021/bi027332w
DO - 10.1021/bi027332w
M3 - Article
C2 - 12795611
AN - SCOPUS:0038131065
SN - 0006-2960
VL - 42
SP - 7152
EP - 7161
JO - Biochemistry
JF - Biochemistry
IS - 23
ER -