Kinetic and magnetic resonance studies of active-site mutants of staphylococcal nuclease: factors contributing to catalysis

Abstract

To determine the origin of the overall approximately 10(16)-fold rate enhancement of DNA hydrolysis catalyzed by staphylococcal nuclease, the effects of single mutations that alter the amino acid residue at each of the essential positions Asp-21, Asp-40, Thr-41, Arg-35, and Arg-87 have been examined. Metal ion and substrate analogue binding were quantitated by EPR, by the paramagnetic effects of Mn2+ on 1/T1 of water protons, and by fluorescence titrations, yielding the six dissociation constants of the ternary enzyme-Mn2+-3',5'-pdTp and enzyme-Ca2+-3',5'-pdTp complexes. The kinetic parameters kcat, KACa, KMCa, KSDNA, KMDNA, and KIMn were determined by monitoring the rate of DNA hydrolysis. By thermodynamic and kinetic criteria, Mn2+ binds tightly to the Ca2+ binding site of the enzyme but is at least 36,000-fold less effective than Ca2+ in activating the enzyme. Alterations of the liganding residues in the D40G, D40E, T41P, D21E, and D21Y mutants generally weaken the binding of Ca2+ less than or equal to 12.7-fold and of Mn2+ less than or equal to 5.4-fold, exert little effect on the KSDNA or KMDNA (less than or equal to 3.2-fold), and raise the affinity of the enzyme and its metal complexes for 3',5'-pdTp by factors less than or equal to 13.5-fold. Small changes in the ligand geometry are also reflected in the Mn2+ complexes of the liganding mutants (i.e., those in which the metal-liganding amino acids have been altered) by decreases in the electron-spin relaxation time of Mn2+. Inhibitory effects on kcat are noted in all of the liganding mutants with D40E, D40G, T41P, D21E, and D21Y showing 12-, 30-, 37-, 1500-, and greater than or equal to 29,000-fold reductions, respectively. The greater than or equal 10(3)-fold larger inhibitory effects on kcat of enlarging Asp-21 as compared to enlarging Asp-40 are ascribed to the displacement of the adjacent water molecule which attacks the phosphodiester. Mutations of each of the essential Arg residues to Gly (R35G and R87G) reduce kcat by factors greater than or equal to 35,000 but weaken metal binding less than or equal to 9-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Native Recombinant Cyclophilins A, B, and C Degrade DNA Independently of Peptidylprolyl cis-trans-Isomerase Activity

“…This highly efficient enzyme (which has a turnover rate of 95 s Ϫ1 ; Ref. 57) degrades DNA approximately 1000 times faster than CypC. CypC, in turn, displays a nuclease activity approximately 100 times greater than CypA and CypB.…”

Directing Sequence-specific Proteolysis to New Targets

“…Conversion of V max into k cat allows direct comparison with the background rate of phosphodiester hydrolysis. The lowest k cat we calculate for any variant, 0.1 s Ϫ1 , was over 12 orders of magnitude greater than the pseudo-first order rate constant determined for the uncatalyzed reaction, 5.7 (10) Ϫ14 s Ϫ1 (23,(33)(34)(35). Retention of substantial catalytic activity suggests that most of the structural features necessary for catalysis are present in the variants and that their overall structure is similar to the wild-type enzyme.…”

“…V max is used to characterize the kinetics of SNase catalysis because of the heterogeneous nature of the DNA substrate, but it can be converted into an approximate k cat value assuming the molecular weight of an average substrate tetranucleotide to be 1400 and a change in absorbance of 0.3 A 260 for complete hydrolysis of 50 g/l DNA (22,23). Conversion of V max into k cat allows direct comparison with the background rate of phosphodiester hydrolysis.…”

“…Errors were calculated as described (21). V max is usually reported rather than k cat because of the heterogeneous nature of the chromosomal DNA substrate, but for purposes of comparison with the uncatalyzed pseudo-first order rate we assumed an average molecular weight for tetranucleotide substrate of 1400 and a change in absorbance of 0.3 OD for complete hydrolysis of 50 mg/ml DNA (22,23).…”

Protein engineering. The design, synthesis and characterization of factitious proteins

“…The first crop of kinetic and n.m.r. results for mutants at other sites believed to contribute to catalysis emphasizes how much can be learned from the exploitation of well-studied proteins (Serpersu et al, 1987).…”