Putative secondary active site of bovine pancreatic deoxyribonuclease I

Previous structural studies based on the co-crystal of a complex between bovine pancreatic deoxyribonuclease I (bpDNase I) and a double-stranded DNA octamer d(GCGATCGC)(2) have suggested the presence of a putative secondary active site near Ser43. In our present study, several crucial amino acid residues postulated in this putative secondary active site, including Thr14, Ser43, and His44 were selected for site-directed mutagenesis. A series of single, double and triple mutants were thus constructed and tested for their DNase I activity by hyperchromicity assay. Substitution of each or both of Thr14 and Ser43 by alanine results in mutant enzymes retaining 30-70% of WT bpDNase I activity. However, when His44 was replaced by aspartic acid, either in the single, double, or triple mutant, the enzyme activities were drastically decreased to 0.5-5% that of WT bpDNase I. Interestingly, when cysteine was substituted for Thr14 or Ser43, the specific DNase activities of the mutant enzymes were substantially increased by 1.5-100-fold, comparing to their alanine substitution mutant counterparts. Two other more sensitive DNase activity assay method, plasmid scission and zymogram analyses further confirm these observations. These results suggested that His44 may play a critical role in substrate DNA binding in this putative secondary active site, and introduction of sulfhydryl groups at Thr14 and Ser43 may facilitate Mn(2+)-coordination and further contribute to the catalytic activity of bpDNase I.     

Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

We characterized the biochemical functions of the small nonessential (C101-C104) and the large essential (C173-C209) disulfides in bovine pancreatic (bp) DNase using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. We also characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca(2+) protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin, whereas brDNase(F192C/A217C) remained active. With Ca(2+), all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca(2+)-containing buffer. However, when diluted into a Ca(2+)-free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65 degrees C, brDNase(C101A) at 60 degrees C, and brDNase(F192C/A217C) at 73 degrees C, indicating that the small disulfide, albeit not essential for activity, is important for the structural integrity, and that the introduction of a third disulfide can further stabilize the enzyme. When pellets of brDNase(C173A) and brDNase(C209A) in inclusion bodies were dissolved in 6 M guanidine-HCl and then diluted into a Ca(2+)-containing buffer, 10%-18% of the bpDNase activity was restored, suggesting that the "essential" disulfide is not absolutely crucial for enzymatic catalysis. Owing to the structure-based sequence alignment revealing homology between the "nonessential" disulfide of bpDNase and the active-site motif of thioredoxin, we measured 39% of the thioredoxin-like activity for bpDNase based on the rate of insulin precipitation (DeltaA650nm/min). Thus, the disulfides in bpDNase not only play the role of stabilizing the protein molecule but also may engage in biological functions such as the disulfide/dithiol exchange reaction.     

Interdependent folding of the N- and C-terminal domains defines the cooperative folding of alpha-lytic protease

Alpha-lytic protease (alphaLP) serves as an important model in achieving a quantitative and physical understanding of protein folding reactions. Synthesized as a pro-protease, alphaLP belongs to an interesting class of proteins that require pro regions to facilitate their proper folding. alphaLP's pro region (Pro) acts as a potent folding catalyst for the protease, accelerating alphaLP folding to its native conformation nearly 10(10)-fold. Structural and mutational studies suggested that Pro's considerable foldase activity is directed toward structuring the alphaLP C-terminal domain (CalphaLP), a seemingly folding-impaired domain, which is believed to contribute significantly to the high-energy folding and unfolding transition states of alphaLP. Pro-mediated nucleation of alphaLP folding within CalphaLP was hypothesized to subsequently enable the alphaLP N-terminal domain (NalphaLP) to dock and fold, completing the formation of native protease. In this paper, we find that ternary folding reactions of Pro and noncovalent NalphaLP and CalphaLP domains are unaffected by the order in which the components are added or by the relative concentrations of the alphaLP domains, indicating that neither discrete CalphaLP structuring nor docking of the two alphaLP domains is involved in the folding transition state. Instead, the rate-limiting step of these folding reactions appears to be a slow and concerted rearrangement of the NalphaLP and CalphaLP domains to form active protease. This cooperative and interdependent folding of both protease domains defines the large alphaLP folding barrier and is an apparent extension of the highly cooperative alphaLP unfolding transition that imparts the protease with remarkable kinetic stability and functional longevity.     

Structure and function of bovine pancreatic deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (bpDNase), the first DNase discovered, is the best characterized among various types of DNase. A catalytic mechanism has been suggested based on the X-ray structure of the bpDNase-octamer complex. In this review, we will focus on three aspects: 1) the distinctive functions of the two structural calcium atoms; 2) the biological functions of the two disulfides; and 3) the involvement of the N- and C-terminal fragments in the enzyme folding for activity.     

Simple purification and properties of bovine pancreatic deoxyribonuclease I

A simple purification method for pancreatic deoxyribonuclease I (DNase I) [EC 3.1.4.3] was developed by utilizing the technique of isoelectric focusing. The active protein was resolved in to at least four forms with different isoelectric points; the major components a, b, and c had isoelectric points at pH 5.2, 4.9, and 4.8, respectively, and that of the minor component d was at 4.7. The four components (a, b, c, and d) exhibited peaks similar to those observed by Salnikow et al. after phosphocellulose chromatography (A, B, C, and D). The four components were all free from RNase and protease activities and were very stable at 0-2 degrees C for at least four weeks. Further, each of the four peaks exhibited a single protein band after polyacrylamide electrophoresis. DNase I-a antibody was prepared; it was very specific for DNase I and precipitated with the other components (b, c, and d). The mode of endonucleolytic action of pancreatic DNase I-a purified from Worthington DP grade DNase I was investigated. The sedimentation patterns in neutral sucrose gradients of digest of circular duplex DNA in an early stage of hydrolysis suggested that DNase I produces single strand scissions in the initial attack in the presence of divalent metal ions.     

Crystallization and preliminary crystallographic data of bovine pancreatic deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I has been crystallized using the method of precipitation by polyethylene glycol 6000. The crystals diffract to 1.8 Å and belong to space group C2 with cell dimensions: $\text{a = 131.6}\text{A}\text{?}\text{, b = 54.9}\text{A}\text{?}\text{, c = 38.4}\text{A}\text{?}\text{, β = 91.4 °}$ and one molecule per asymmetric unit.     

Observations on the carbohydrate moiety of bovine pancreatic deoxyribonuclease A

The carbohydrate side chain of bovine pancreatic deoxyribonuclease A, which is attached to asparagine residue 18, contains two residues of N-acetylglucosamine proximal to the peptide chain followed by a variable number of mannose residues (4–10). The oligosaccharide structure bears a similarity to that in bovine pancreatic ribonuclease B. The present sequence studies have made use of α-mannosidase chromatographically purified from jack bean meal.     

Catalysis of the oxidative folding of bovine pancreatic ribonuclease A by protein disulfide isomerase

The major oxidative folding pathways of bovine pancreatic ribonuclease A at pH 8.0 and 25 degrees C involve a pre-equilibrium steady state among ensembles of intermediates with zero, one, two, three and four disulfide bonds. The rate-determining steps are the reshuffling of the unstructured three-disulfide ensemble to two native-like three-disulfide species, des-[65-72] and des-[40-95], that convert to the native structure during oxidative formation of the fourth disulfide bond. Under the same regeneration conditions, with oxidized and reduced DTT, used previously for kinetic oxidative-folding studies of this protein, the addition of 4 microM protein disulfide isomerase (PDI) was found to lead to catalysis of each disulfide-formation step, including the rate-limiting rearrangement steps in which the native-like intermediates des-[65-72] and des-[40-95] are formed. The changes in the distribution of intermediates were also determined in the presence and absence of PDI at three different temperatures (with the DTT redox system) as well as at 25 degrees C (with the glutathione redox system). The results indicate that the acceleration of the formation of native protein by PDI, which we observed earlier, is due to PDI catalysis of each of the intermediate steps without changing the overall pathways or folding mechanism.     

The interaction of bovine pancreatic deoxyribonuclease I and skeletal muscle actin

The rate of exchange of actin-bound nucleotide is decreased by a factor of about 20 when actin is complexed with DNAase I without affecting the binding constant of calcium for actin. Binding constants of DNAase I to monomeric and filamentous actin were determined to be 5 X 10(8) M-1 and 1.2 X 10(4) M-1 respectively. The depolymerisation of F-actin by DNAase I appears to be due to a shift in the G-F equilibrium of actin by DNAase I. Inhibition of the DNA-degrading activity of DNAase I by G-actin is of the partially competitive type.     

Hydrolysis of p-nitrophenyl phenylphosphonate catalysed by bovine pancreatic deoxyribonuclease.

The ability of bovine pancreatic DNAase to hydrolyse the synthetic substrate p-nitrophenyl phenylphosphonate (NPPP) is intrinsic and is not due to the contamination of the DNAase preparation by nonspecific phosphodiesterases because the activities of DNA and NPPP hydrolysis are co-eluted from a DEAE-cellulose column with use of the Ca2+-affinity elution method and because the two activities are decreased simultaneously when the purified enzyme is treated with Cu2+/iodoacetate, an active-site-labelling agent for DNAase. NPPP hydrolysis is facilitated by the metal ion-DNAase. At relatively high Na+ concentrations, where the metal ion-DNA interaction is weak, DNA hydrolysis is also facilitated by the metal ion-DNAase. With NPPP as substrate the Michaelis constants are Km 3.7 mM for Mn2+ and Km 49 mM for Mg2+ in 0.2 M-Tris/HCl buffer, pH 7.2. Ca2+ competes with Mn2+, with Ki 64 mM. Free Cu2+ ions non-competitively inhibit DNAase-catalysed DNA or NPPP hydrolysis in the presence of Mn2+ or Mg2+ and the inhibition is not relieved by Ca2+. The affinity of Cu2+ for free DNAase is higher than that for Mn2+-DNAase. Mn2+ is not bound to DNAase via a simple ionic interaction, as Mn2+ remains bound in the presence of relatively high Na+ concentrations and induces a near-u.v. difference absorption spectrum. The kinetics of NPPP hydrolysis catalysed by Mn2+-DNAase are sigmoidal. From the Hill equation, h = 2.0 is obtained, suggesting that more than two NPPP molecules are bound per molecule of DNAase with a certain amount of co-operativity. Because DNAase in solution is a monomer with a single catalytic site, the multiple NPPP molecules on a single protein molecule are probably in one location, resulting in a co-operative interaction that may resemble that in the stacked base-pairs of double-helical DNA.     

The distinctive functions of the two structural calcium atoms in bovine pancreatic deoxyribonuclease

The two amino acid residues, Asp 99 and Asp 201, involved in the coordination of the two calcium atoms in the X-ray structure of bovine pancreatic (bp) DNase, were individually changed by site-directed mutagenesis. The two altered proteins, brDNase(D99A) and brDNase(D201A) were expressed in Escherichia coli and purified by anion exchange chromatography. Equilibrium dialysis showed that mutation destroyed one Ca(2+)-binding site each in brDNase(D99A) and brDNase(D201A). Compared with bpDNase, the Vmax value for brDNase(D99A) remained unchanged and that for brDNase(D201A) was decreased, whereas the K(m) values for the two variants were increased two- to threefold when the DNA hydrolytic hyperchromicity assay was used. Like bpDNase, brDNase(D99A) was able to make double scission on duplex DNA with Mg(2+) plus Ca(2+) and was effectively protected by Ca(2+) from the trypsin inactivation. But under the same conditions, brDNase(D201A) lost the double-scission ability and was not protected by Ca(2+). Nevertheless, the two variant proteins retained the characteristics of the Ca(2+)-induced conformational changes and the Ca(2+) protection against the beta-mercaptoethanol disruption of the essential disulfide bond, suggesting that other weaker Ca(2+)-binding sites not found in the X-ray structure were responsible for these properties. Therefore, the two structural calcium atoms are not for maintaining the overall conformation of the active DNase, as it has been indicated in the X-ray analysis, but rather play the role in the fine-tuning of the DNase activity.     

Structure-function analysis of PrsA reveals roles for the parvulin-like and flanking N- and C-terminal domains in protein folding and secretion in Bacillus subtilis

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.     

Probing the catalytic mechanism of bovine pancreatic deoxyribonuclease I by chemical rescue

Previous structural and mutational studies of bovine pancreatic deoxyribonuclease I (bpDNase I) have demonstrated that the active site His134 and His252 played critical roles in catalysis. In our present study, mutations of these two His residues to Gln, Ala or Gly reduced the DNase activity by a factor of four to five orders of magnitude. When imidazole or primary amines were added exogenously to the Ala or Gly mutants, the residual DNase activities were substantially increased by 60-120-fold. The rescue with imidazole was pH- and concentration-dependent. The pH-activity profiles showed nearly bell-shaped curves, with the maximum activity enhancement for H134A at pH 6.0 and that for H252A at pH 7.5. These findings indicated that the protonated form of imidazole was responsible for the rescue in H134A, and the unprotonated form was for that in H252A, prompting us to assign unambiguously the roles for His134 as a general acid, and His252 as a general base, in bpDNase I catalysis.