Kinetics and equilibria in ligand binding by nitrophorins 1-4: evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap

Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 microM(-)(1) s(-)(1)) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows ( approximately 50 s(-)(1)), and the stabilized final complexes are resistant to autoreduction (E degrees = +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s(-)(1)), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s(-)(1)). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.     

An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3

We have examined the enzymatic activity of an uncleavable procaspase-3 mutant (D9A/D28A/D175A), which contains the wild-type catalytic residues in the active site. The results are compared to those for the mature caspase-3. Although at pH 7.5 and 25 degrees C the K(m) values are similar, the catalytic efficiency (k(cat)) is approximately 130-fold lower in the zymogen. The mature caspase-3 demonstrates a maximum activity at pH 7.4, whereas the maximum activity of procaspase-3 occurs at pH 8.3. The pK(a) values of both catalytic groups, H121 and C163, are shifted to higher pH for procaspase-3. We developed limited proteolysis assays using trypsin and V8 proteases, and we show that these assays allow the examination of amino acids in three of five active site loops. In addition, we examined the fluorescence emission of the two tryptophanyl residues in the active site over the pH range of 2.5-9 as well as the response to several quenching agents. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle among loops L4, L2, and L2'. The pK(a) values of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate binding pocket, does not appear to be unraveled and solvent-exposed, even at lower pH.     

Spectroscopic and functional characterization of nitrophorin 7 from the blood-feeding insect Rhodnius prolixus reveals an important role of its isoform-specific N-terminus for proper protein function

Nitrophorins (NPs) are a class of NO-transporting and histamine-sequestering heme b proteins that occur in the saliva of the bloodsucking insect Rhodnius prolixus. A detailed study of the newly described member, NP7, is presented herein. NO association constants for NP7 [KIII(eq)(NO)] reveal a drastic change when the pH is varied from 5.5 (reflecting the insect's saliva) to slightly above plasma pH (7.5) (>10(9) M-1 --> 4.0 x 10(6) M-1); thus, the protein promotes the storage of NO in the insect's saliva and its release inside the victim's tissues. In contrast to the other nitrophorins, NP1-4, histamine sequestering cannot be accomplished in vivo due to the low binding constant [KIII(eq)(histamine)] of 10(5) M-1 compared to the histamine concentration of 1-10 x 10(-9) M in the blood. A major part of this study deals with the N-terminus, 1Leu-Pro-Gly-Glu-Cys5 of NP7, which is not found in NP1-4. Since NP7 has not been isolated from the insects but was recognized in a cDNA library instead, the N-terminal site of signal peptidase cleavage upon protein secretion was predicted by the program SIGNALP [Andersen, J. F., Gudderra, N. P., Francischetti, I. M. B., Valenzuela, J. G., and Ribeiro, J. M. C. (2004) Biochemistry 43, 6987-6994]. In marked contrast to wild-type NP7, NP7(Delta1-3) exhibits a very high NO affinity at pH 7.5 [KIII(eq)(NO) approximately 10(9) M-1], suggesting that the release of NO in the plasma cannot efficiently be accomplished by the truncated form. Comparison of the reduction potentials of both constructs by spectroelectrochemistry revealed an average increase of +85 mV for various distal ligands bound to the heme iron when the 1Leu-Pro-Gly3 peptide was removed. However, 1H NMR and EPR spectroscopy show that the electronic properties of the FeIII cofactor are similar in both wild-type NP7 and NP7(Delta1-3). Further, thermal denaturation that revealed a higher stability of wild-type NP7 compared to NP7(Delta1-3), in combination with a homology model based on the NP2 crystal structure (rmsd = 0.39 A), suggests that interaction of the 1Leu-Pro-Gly3 peptide with the A-B and/or G-H loops is key for proper protein function.     

Breaking the proximal Fe(II)-N(His) bond in heme proteins through local structural tension: lessons from the heme b proteins nitrophorin 4, nitrophorin 7, and related site-directed mutant proteins

The factors leading to the breakage of the proximal iron-histidine bond in the ferroheme protein soluble guanylate cyclase (sGC) are still a matter of debate. This event is a key mechanism in the sensing of NO that leads to the production of the second-messenger molecule cGMP. Surprisingly, in the heme protein nitrophorin 7 (NP7), we noticed by UV-vis absorbance spectroscopy and resonance Raman spectroscopy that heme reduction leads to a loss of the proximal histidine coordination, which is not observed for the other isoproteins (NP1-4). Structural considerations led to the generation and spectroscopic investigation of site-directed mutants NP7(E27V), NP7(E27Q), NP4(D70A), and NP2(V24E). Spectroscopic investigation of these proteins shows that the spatial arrangement of residues Glu27, Phe43, and His60 in the proximal heme pocket of NP7 is the reason for the weakened Fe(II)-His60 bond through steric demand. Spectroscopic investigation of the sample of NP7 reconstituted with 2,4-dimethyldeuterohemin ("symmetric heme") demonstrated that the heme vinyl substituents are also responsible. Whereas the breaking of the iron-histidine bond is rarely seen among unliganded ferroheme proteins, the breakage of the Fe(II)-His bond upon binding of NO to the sixth coordination site is sometimes observed because of the negative trans effect of NO. However, it is still rare among the heme proteins, which is in contrast to the case for trans liganded nitrosyl model hemes. Thus, the question of which factors determine the Fe(II)-His bond labilization in proteins arises. Surprisingly, mutant NP2(V24E) turned out to be particularly similar in behavior to sGC; i.e., the Fe(II)-His bond is sensitive to breakage upon NO binding, whereas the unliganded form binds the proximal His at neutral pH. To the best of our knowledge, NP2(V24E) is the first example in which the ability to use the His-on ? His-off switch was engineered into a heme protein by site-directed mutagenesis other than the proximal His itself. Steric tension is, therefore, introduced as a potential structural determinant for proximal Fe(II)-His bond breakage in heme proteins.     

An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis.

The nucleocapsid protein (NP) of Sendai virus encapsidates the genome RNA, forming a helical nucleocapsid which is the template for RNA synthesis by the viral RNA polymerase. The NP protein is thought to have both structural and functional roles, since it is an essential component of the NP0-P (P, phosphoprotein), NP-NP, nucleocapsid-polymerase, and RNA-NP complexes required during viral RNA replication. To identify domains in the NP protein, mutants were constructed by using clustered charge-to-alanine mutagenesis in a highly charged region from amino acids 107 to 129. Each of the mutants supported RNA encapsidation in vitro. The product nucleocapsids formed with three mutants, NP114, NP121, and NP126, however, did not serve as templates for further amplification in vivo, while NP107, NP108, and NP111 were nearly like wild-type NP in vivo. This template defect in the NP mutants from amino acids 114 to 129 was not due to a lack of NP0-P, NP-NP, or nucleocapsid-polymerase complex formation, since these interactions were normal in these mutants. We propose that amino acids 114 to 129 of the NP protein are required for the nucleocapsid to function as a template in viral genome replication.     

Insertion of an h-bonding residue into the distal pocket of the ferriheme protein nitrophorin 4: effect on nitriteiron coordination and nitrite disproportionation

Heme proteins are important entities for the metabolism of nitrite. Inspection of the structural features of the reported hemoprotein?nitrite crystal structures reveals that, except for nitrophorin 4 (NP4), H-bonding to the nitrite ligand is accomplished via histidine or arginine residues. These H-bonds probably play an important role for the nitrite coordination and/or reactivities. In nitrophorins, which catalyze the nitrite disproportionation reaction, such a residue is missing. Here, we report on the L130R mutant of the NP isoprotein NP4 that provides the Arg130 residue as part of the flexible G?H loop as a potential H-bonding residue in the distal heme pocket. Similar to the wild-type protein, nitrite remains N-bonded in the crystal structure of NP4(L130R). However, spectroscopic investigations show that, in solution, a second ligand-rotational orientation exists, which is in fast-exchange equilibrium with the normal, parallel ligand orientation. Moreover, the nitrite disproportionation is inhibited in NP4(L130R). Comparison with another, also less active mutant NP4(D30N) suggests that the displacement of H(2) O molecules from the heme cavity prevents the proton donation pathway through Asp30.     

Effect of the N-terminus on heme cavity structure, ligand equilibrium, rate constants, and reduction potentials of nitrophorin 2 from Rhodnius prolixus

The D1A mutant of recombinant NP2 has been prepared and shown to have the expression-initiation methionine-0 cleaved during expression in E. coli, as is the case for recombinant NP4, where Ala is the first amino acid for the recombinant protein as well as for the mature native protein. The heme substituent 1H NMR chemical shifts of NP2-D1A and those of its imidazole, N-methylimidazole, and cyanide complexes are rather different from those of NP2-M0D1. This difference is likely due to the much smaller size of the N-terminal amino acid (A) of NP2-D1A, which allows the formation of the closed loop form of this protein, as it does for NP4 (Weichsel, A., Andersen, J. F., Roberts, S. A., and Montfort, W. R. (2000) Nature Struct. Biol. 7, 551-554). The ratio of the two hemin rotational isomers A and B is different for the two proteins, and the rate at which the A:B ratio reaches equilibrium is strikingly different (NP2-M0D1 t1/2 for heme rotation approximately 2 h, NP2-D1A t1/2 approximately 43 h). This difference is consistent with the high stability of the closed loop form of the NP2-D1A protein and infrequent opening of the loops that could allow heme to at least partially exit the binding pocket in order to rotate about its alpha,gamma-meso axis. Consistent with this, the rates of histamine binding and release to/from NP2-D1A are significantly slower than those for NP2-M0D1 at pH 7.5. This work suggests that care must be taken in interpreting data obtained from proteins that carry the expression-initiation M0.     

Structural changes accompanying pH-induced dissociation of the beta-lactoglobulin dimer

We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and to characterize the backbone dynamics, of a recombinant version of bovine beta-lactoglobulin (variant A) at pH 2. 6, where the protein is a monomer. The structure of this low-pH form of beta-lactoglobulin is very similar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2 (crystal) structure, calculated for backbone atoms of residues 6-160, is approximately 1.3 A. Differences arise from the orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn alpha-helix. The hydrophobic cavity within the calyx is retained at low pH. The E-F loop (residues 85-90), which moves to occlude the opening of the cavity over the pH range 7.2-6.2, is in the "closed" position at pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of (15)N T(1)s and T(2)s and (1)H-(15)N heteronuclear NOEs at pH 2.6 and 37 degrees C. Although the residues of the E-F loop (residues 86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well defined by the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Several residues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T(1) to T(2), consistent with conformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3D structure of beta-lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity.     

NMR studies of nitrophorin distal pocket side chain effects on the heme orientation and seating of NP2 as compared to NP1

The nitrophorins (NP) of the adult blood-sucking insect Rhodnius prolixus fall into two pairs based on sequence identity (NP1,4 (90%) and NP2,3 (79%)), which differ significantly in the size of side chains of residues which contact the heme. These residues include those in the distal pocket of NP2 (I120) and NP1 (T121) and the “belt” that surrounds the heme of NP2 (S40, F42), and NP1(A42, L44). To determine the importance of these residues and others conserved or very similar for the two pairs, including L122(123), L132(133), appropriate mutants of NP2 and NP1 have been prepared and studied by ¹H NMR spectroscopy. Wild-type NP2 has heme orientation ratio (A:B) of 1:8 at equilibrium, while wild-type NP1 has A:B ~ 1:1 at equilibrium. Another difference between NP2 and NP1 is in the heme seating with regard to His57(59). It is found that among the distal pocket residues investigated, the residue most responsible for heme orientation and seating is I120(T121). F42(L44) and L106(F107) may also be important, but must be investigated in greater detail.     

Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway

The role of the polymorphism Met or Val in position 129 in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. However, little is known about the molecular background to this phenomenon. We investigated herein the conformational stability, amyloid fibrillation kinetics, and seeding propensity of different 129 mutants, located in β-strand 1 of PrP (Met(129) (WT), M129A, M129V, M129L, M129W, M129P, M129E, M129K, and M129C) in HuPrP(90-231). The mutations M129V, M129L, M129K, and M129C did not affect stability (midpoints of thermal denaturation, T(m) = 65-66 °C), whereas the mutants M129A and M129E and the largest side chain M129W were destabilized by 3-4 °C. The most destabilizing substitution was M129P, which lowered the T(m) by 7.2 °C. All mutants, except for M129C, formed amyloid-like fibrils within hours during fibril formation under near physiological conditions. Fibril-forming mutants showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants M129A, M129V, and M129L resembling the wild type. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (M129W), charge (M129E, M129K), or rotational constraint (M129P), the fibrillation was impeded. M129C did not form ThT/Congo red-positive fibrils, and non-reducing SDS-PAGE of M129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 min after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness toward recruitment into the amyloid state.     

Trans membrane domain IV is involved in ion transport activity and pH regulation of the NhaA-Na(+)/H(+) antiporter of Escherichia coli.

We have previously shown that the activity of NhaA is regulated by pH and found mutations that affect dramatically the pH dependence of the rate but not the K(m) (for Na(+) and Li(+)) of NhaA. In the present work, we found that helix IV is involved both in ion translocation as well as in pH regulation of NhaA. Two novel types of NhaA mutants were found clustered in trans membrane segment (TMS) IV: One type (D133C, T132C, and P129L) affects the apparent K(m) of NhaA to the cations with no significant effect on the pH profile of the antiporter; no shift of the pH profile was found when the activity of these mutants was measured at saturating Na(+) concentration. In contrast, the other type of mutations (A127V and A127T) was found to affect both the K(m) and the pH dependence of the rate of NhaA whether tested at saturating Na(+) concentration or not. These results imply that residues involved in the ion translocation of NhaA may (A127) or may not (D133, T132, and P129) overlap with those affecting the pH response of the antiporter. All mutants cluster in the N-terminal half of the putative alpha-helix IV, one type on one face, the other on the opposite. Cys accessibility test demonstrated that although D133C is located in the middle of TMS IV, it is inhibited by N-ethylmaleimide and is exposed to the cytoplasm.     

The catalytic properties of Thermus thermophilus SG0.5JP17-16 laccase were regulated by the conformational dynamics of pocket loop 6

BackgroundLaccase is one member of the blue multicopper oxidase family. It can catalyze the oxidation of various substrates. The Thermus thermophilus SG0.5JP17-16 laccase (lacTT) is thermostable, pH-stable, and high tolerance to halides, and can decolorize the synthetic dyes. In lacTT, the function of the loop 6 constructing the substrate-binding pocket wasn't clear.MethodsThe residues Asp394 and Asp396 located in loop 6, and were used to probe how the loop 6 influenced catalytic properties of the laccase. Site-directed mutagenesis was performed for two amino acids. Kinetic assay was utilized to characterize the catalytic efficiency of mutants. Mutants with different catalytic activities were used to decolorize the synthetic dyes to clarify the relationship between the catalytic efficiency and dye decolorization. Redox potential, structural and spectral analyses were performed to explain the differences in laccase activity between wild type and mutant enzymes.ResultsD394M, D394E and D394R mutants with the lower laccase activity displayed a decreased decolorization efficiency, while D396A, D396M and D396E mutant enzymes with higher catalytic efficiency decolorized the synthetic dye more efficiently than the wild type enzyme.ConclusionsThe pocket loop 6 might experience a conformational dynamics. The D394 residue controlled this conformation change by amino acid interaction networks containing the D396 residue at the entrance of substrate channel.General significancesThese studies may provide clues to improve the activity of the laccase for the better use in industrial applications, and/or contribute to further understanding the mechanism of laccase oxidation on the substrate.     

Structural determinants of factor IX(a) binding in nitrophorin 2, a lipocalin inhibitor of the intrinsic coagulation pathway

Nitrophorin 2 (NP2) is a salivary lipocalin from Rhodnius prolixus that binds with coagulation factors IX (fIX) and IXa (fIXa). Binding of NP2 with fIXa results in potent inhibition of the intrinsic factor Xase complex. A panel of site-directed surface mutants of NP2 was generated to locate determinants of high affinity fIX(a) binding. The locations of the mutations were based on comparisons with the related, but less potent, inhibitor nitrophorin 3 (NP3). Three point mutants (K21A, K92A, and V94A) were found that clearly reduced the inhibitory potency as measured by the activity of a reconstituted factor Xase system. Binding of NP2 with fIXa and fIX as measured by surface plasmon resonance and isothermal titration calorimetry was reduced in a similar manner. Of the three mutants, two (K92A and V94A) were located on the loop connecting beta-strands E and F of the lipocalin beta-barrel. The largest changes were seen with the K92A mutation, which lies at the apex of the loop, with a smaller effect being seen with mutation of Val(94). Combination of four E-F loop mutations (K92A, A93K, V94A, E97A) in a single mutant reduced the inhibitory potency and binding to levels similar to those seen with NP3 without affecting heme or histamine binding.     

Restricted backbone conformational and motional flexibilities of loops containing peptidyl-proline bonds dominate the enzyme activity of staphylococcal nuclease

The role of cis-trans isomerizations of peptidyl-proline bonds in the enzyme activity of staphylococcal nuclease (SNase) was examined by mutation of proline residues. The proline-free SNase ([Pro-]SNase), namely, P11A/P31A/P42A/P47T/P56A/P117G-mutant SNase, was adopted for elucidating the correlation between the nuclease activity and the backbone conformational and dynamic states of SNase. The 3D solution structure of [Pro-]SNase has been determined by heteronuclear NMR experiments. Comparing the structure of [Pro-]SNase with the structure of SNase revealed the conformational differences between the two proteins. In the structure of [Pro-]SNase, conformational rearrangements were observed for the loop of residues Ala112-His121 containing a trans Lys116-Gly117 peptide bond and for the C-terminal alpha-helical loop of residues Leu137-Glu142. Mutation of proline at position 117 also caused the conformational rearrangement of the p-loop (Asp77-Leu89), which is remote from the Ala112-His121 loop. The Ala112-His121 loop and p-loop are placed closer to each other in [Pro-]SNase than in SNase. The backbone dynamic features of the omega-loop (Pro42-Pro56) of SNase are different from those of [Pro-]SNase. The backbone of the omega-loop exhibits restricted flexibility with slow conformational exchange motions in SNase, but is highly flexible in [Pro-]SNase. The analysis indicates that the restrained backbone conformation of the Ala112-His121 loop and restricted flexibility of the omega-loop are two dominant factors determining the enzyme activity of SNase. Of the two factors, the former is correlated with the strained cis Lys116-Pro117 peptide bond and the latter is correlated with the cis-trans isomerizations of the His46-Pro47 peptide bond.     

VP3 G–H环中氨基酸突变对O型口蹄疫病毒生物学特性的影响

【目的】为了研究O型口蹄疫病毒VP3G–H环中氨基酸突变对其生物学特性的影响。【方法】借助口蹄疫病毒反向遗传操作技术平台拯救出2株定点突变体rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)。进行蚀斑形成试验、一步生长曲线的绘制、TCID_(50)和LD_(50)的测定、间接免疫荧光与激光共聚焦显微镜检测。【结果】结果显示,与骨架病毒rHN相比,虽然rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)对BHK-21细胞的感染性及其蚀斑表型和复制动力学无显著性差异;但rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)对乳鼠的致病力明显减弱,且均获得了小窝蛋白介导侵染CHO-K1细胞的能力。【结论】VP3上第3174位特征性氨基酸突变影响O型口蹄疫病毒感染宿主细胞的毒力及其内吞作用路径,这有助于我们认知VP3 G–H环在口蹄疫病毒粒子立体空间构象中潜在的作用。     

Mechanistic analysis of A46V,H57Y,and D129N in pyrazinamidase associated with pyrazinamide resistance

Pyrazinamide (PZA) is a component of first-line drugs, active against latent Mycobacterium tuberculosis (MTB) isolates. The prodrug is activated into the active form, pyrazinoic acid (POA) via pncA gene-encoded pyrazinamidase (PZase). Mutations in pncA have been reported, most commonly responsible for PZA-resistance in more than 70% of the resistant cases. In our previous study, we detected many mutations in PZase among PZA-resistance MTB isolates including A46V, H71Y, and D129N. The current study was aimed to investigate the molecular mechanism of PZA-resistance behind mutants (MTs) A46V, H71Y, and D129N in comparison with the wild type (WT) through molecular dynamic (MD) simulation. MTB positive samples were subjected to PZA drug susceptibility testing (DST) against critical concentration (100ug/ml). The resistant samples were subjected to pncA sequencing. Thirty-six various mutations have been observed in the coding region of pncA of PZA-resistant isolates (GenBank accession No. MH461111) including A46V, H71Y, and D129N. The post-simulation analysis revealed a significant variation in MTs structural dynamics as compared to the WT. Root means square deviations (RMSD) and Root means square fluctuation (RMSF) has been found in variation between WT and MTs. Folding effect and pocket volume were altered in MTs when compared with WT. Geometric matching supports the effect of mutation A46V, H71Y, and D129N on PZase structure that may have an insight effect on PZase dynamics, making them vulnerable to convert pro-PZA into active form, POA. In conclusion, the current analyses will provide useful information behind PZA-resistance for better management of drug-resistant TB.     

ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility

Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar approximately 3-fold reduction in both microtubule gliding velocity and microtubule-stimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.     

Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (k_cat/K_m) compared to wild-type CA130. Their K_m values were decreased by ca. 50%, and the k_cat values increased to 14.4 and 16.9 s⁻¹, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.