共查询到20条相似文献,搜索用时 31 毫秒
1.
Giuseppe Rulli Marcel Heidlindemann Albrecht Berkessel Werner Hummel Harald Gröger 《Journal of biotechnology》2013
The alcohol dehydrogenases (ADHs) from Lactobacillus kefir and Rhodococcus sp., which earlier turned out to be suitable for a chemoenzymatic one-pot synthesis with organocatalysts, were immobilized with their cofactors on a commercially available superabsorber based on a literature known protocol. The use of the immobilized ADH from L. kefir in the reduction of acetophenone as a model substrate led to high conversion (>95%) in the first reaction cycle, followed by a slight decrease of conversion in the second reaction cycle. A comparable result was obtained when no cofactor was added although a water rich reaction media was used. The immobilized ADHs also turned out to be suitable catalysts for the diastereoselective reduction of an organocatalytically prepared enantiomerically enriched aldol adduct, leading to high conversion, diastereomeric ratio and enantioselectivity for the resulting 1,3-diols. However, at a lower catalyst and cofactor amount being still sufficient for biotransformations with “free” enzymes the immobilized ADH only showed high conversion and >99% ee for the first reaction cycle whereas a strong decrease of conversion was observed already in the second reaction cycle, thus indicating a significant leaching effect of catalyst and/or cofactor. 相似文献
2.
UDP-galactose 4′-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD+ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia. 相似文献
3.
Thomas J. McCorvie Ying Liu Andrew Frazer Tyler J. Gleason Judith L. Fridovich-Keil David J. Timson 《生物化学与生物物理学报:疾病的分子基础》2012,1822(10):1516-1526
Deficiency of UDP-galactose 4′-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and no ability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD+. p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two α-helices that contain residues that interact with NAD+. These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation. 相似文献
4.
David A. Six Bliss Lambert Christian R.H. Raetz William T. Doerrler 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2012,1821(7):989-993
We previously described enrichment of conditional Escherichia coli msbA mutants defective in lipopolysaccharide export using Ludox density gradients (Doerrler WT (2007) Appl Environ Microbiol 73; 7992–7996). Here, we use this approach to isolate and characterize temperature-sensitive lpxL mutants. LpxL is a late acyltransferase of the pathway of lipid A biosynthesis (The Raetz Pathway). Sequencing the lpxL gene from the mutants revealed the presence of both missense and nonsense mutations. The missense mutations include several in close proximity to the enzyme's active site or conserved residues (E137K, H132Y, G168D). These data demonstrate that Ludox gradients can be used to efficiently isolate conditional E. coli mutants with defects in lipopolysaccharide biosynthesis and provide insight into the enzymatic mechanism of LpxL. 相似文献
5.
Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme''s thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme''s specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described. 相似文献
6.
The glycosyltransferase UGT85H2 from Medicago truncatula catalyzes glucosylation of the (iso)flavonoids kaempferol and biochanin A. Structure-based mutagenesis of UGT85H2 was carried out to explore the roles of amino acids involved in substrate binding. Substitution of Ile305 by threonine increased catalytic efficiency 37- or 19-fold with kaempferol or biochanin A as acceptor, respectively. A point mutation V200E also dramatically improved the turnover rate and catalytic efficiency by 15-fold for kaempferol and 54-fold for biochanin A. More interestingly, this single mutation (V200E) conferred reversibility in the glycosyltransfer reaction, indicating that Glu200 is a key determinant for the deglycosylation function. 相似文献
7.
Carlos H Ni?o Nicolás Forero-Baena Luis E Contreras Diana Sánchez-Lancheros Katherine Figarella María H Ramírez 《Memórias do Instituto Oswaldo Cruz》2015,110(7):890-897
The intracellular parasite Trypanosoma cruzi is the aetiological
agent of Chagas disease, a public health concern with an increasing incidence rate.
This increase is due, among other reasons, to the parasite''s drug resistance
mechanisms, which require nicotinamide adenine dinucleotide (NAD+).
Furthermore, this molecule is involved in metabolic and intracellular signalling
processes necessary for the survival of T. cruzi throughout its life
cycle. NAD+ biosynthesis is performed by de novo and
salvage pathways, which converge on the step that is catalysed by the enzyme
nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission
number: 2.7.7.1). The identification of the NMNAT of T.
cruzi is important for the development of future therapeutic strategies
to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for
NMNAT was identified in the genome of T. cruzi. The corresponding
putative protein was analysed by simulating structural models. The ORF was amplified
from genomic DNA by polymerase chain reaction and was further used for the
construction of a corresponding recombinant expression vector. The expressed
recombinant protein was partially purified and its activity was evaluated using
enzymatic assays. These results comprise the first identification of an NMNAT in
T. cruzi using bioinformatics and experimental tools and hence
represent the first step to understanding NAD+ metabolism in these
parasites. 相似文献
8.
《Journal of molecular biology》2021,433(18):167123
Somatic mutations in the PRKACA gene encoding the catalytic α subunit of protein kinase A (PKA-C) are responsible for cortisol-producing adrenocortical adenomas. These benign neoplasms contribute to the development of Cushing's syndrome. The majority of these mutations occur at the interface between the two lobes of PKA-C and interfere with the enzyme's ability to recognize substrates and regulatory (R) subunits, leading to aberrant phosphorylation patterns and activation. Rarely, patients with similar phenotypes carry an allosteric mutation, E31V, located at the C-terminal end of the αA-helix and adjacent to the αC-helix, but structurally distinct from the PKA-C/R subunit interface mutations. Using a combination of solution NMR, thermodynamics, kinetic assays, and molecular dynamics simulations, we show that the E31V allosteric mutation disrupts central communication nodes between the N- and C- lobes of the enzyme as well as nucleotide-substrate binding cooperativity, a hallmark for kinases' substrate fidelity and regulation. For both orthosteric (L205R and W196R) and allosteric (E31V) Cushing’s syndrome mutants, the loss of binding cooperativity is proportional to the density of the intramolecular allosteric network. This structure–activity relationship suggests a possible common mechanism for Cushing's syndrome driving mutations in which decreased nucleotide/substrate binding cooperativity is linked to loss in substrate fidelity and dysfunctional regulation. 相似文献
9.
Yo Sasaki Zachary Margolin Benjamin Borgo James J. Havranek Jeffrey Milbrandt 《The Journal of biological chemistry》2015,290(28):17228-17238
Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD+ biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD+ synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome. 相似文献
10.
The crystal structure of seabream antiquitin in complex with the cofactor NAD(+) was solved at 2.8A resolution. The mouth of the substrate-binding pocket is guarded by two conserved residues, Glu120 and Arg300. To test the role of these two residues, we have prepared the two mutants E120A and R300A. Our model and kinetics data suggest that antiquitin's specificity towards the substrate alpha-aminoadipic semialdehyde is contributed mainly by Glu120 which interacts with the alpha-amino group of the substrate. On the other hand, Arg300 does not have any specific interaction with the alpha-carboxylate group of the substrate, but is important in maintaining the active site conformation. 相似文献
11.
Strafford J Payongsri P Hibbert EG Morris P Batth SS Steadman D Smith ME Ward JM Hailes HC Dalby PA 《Journal of biotechnology》2012,157(1):237-245
We have previously used targeted active-site saturation mutagenesis to identify a number of transketolase single mutants that improved activity towards either glycolaldehyde (GA), or the non-natural substrate propionaldehyde (PA). Here, all attempts to recombine the singles into double mutants led to unexpected losses of specific activity towards both substrates. A typical trade-off occurred between soluble expression levels and specific activity for all single mutants, but many double mutants decreased both properties more severely suggesting a critical loss of protein stability or native folding. Statistical coupling analysis (SCA) of a large multiple sequence alignment revealed a network of nine co-evolved residues that affected all but one double mutant. Such networks maintain important functional properties such as activity, specificity, folding, stability, and solubility and may be rapidly disrupted by introducing one or more non-naturally occurring mutations. To identify variants of this network that would accept and improve upon our best D469 mutants for activity towards PA, we created a library of random single, double and triple mutants across seven of the co-evolved residues, combining our D469 variants with only naturally occurring mutations at the remaining sites. A triple mutant cluster at D469, E498 and R520 was found to behave synergistically for the specific activity towards PA. Protein expression was severely reduced by E498D and improved by R520Q, yet variants containing both mutations led to improved specific activity and enzyme expression, but with loss of solubility and the formation of inclusion bodies. D469S and R520Q combined synergistically to improve kcat 20-fold for PA, more than for any previous transketolase mutant. R520Q also doubled the specific activity of the previously identified D469T to create our most active transketolase mutant to date. Our results show that recombining active-site mutants obtained by saturation mutagenesis can rapidly destabilise critical networks of co-evolved residues, whereas beneficial single mutants can be retained and improved upon by randomly recombining them with natural variants at other positions in the network. 相似文献
12.
Nicotinamide adenine dinucleotides [NAD and NADP with both referred to as NAD(P)] are among the more diffuse redox cofactors. Despite their stereochemical similarity where the only difference is a phosphomonoester on the ribose near the adenine of NADP, they show different biochemical reactivities with NAD behaving as an oxidant and NADP as a reductant. NAD(P)-dependent enzymes generally share a common open α/β fold with few exceptions only recently structurally characterized. This study of the molecular evolution of the NAD(P) binding domains, possible given the large number of known molecular structures, addresses two main questions: 1) can a common fold exist in different biological systems (divergent evolution) and 2) does a relationship exist among similar biological systems that display different folds (convergent evolution)? Both the structures of mono- and dinucleotide binding domains have been classified by cluster analysis based on the similarity evaluated by their main chain Cα superposition. Moreover, the cofactor conformations and the stereochemical characteristics of their pockets have also been classified by analogous methods on the basis of the published tertiary structures. Two primary results appear: 1) the classification of the mononucleotide binding domains is different from that of the dinucleotide binding folds and 2) both divergent and convergent evolutionary pathways can be hypothesized, the latter less frequently observed and less pronounced but nevertheless evident. The generally accepted hypothesis that dinucleotide binding domains have evolved by gene duplication of primordial genes coding for the smaller mononucleotide binding domains is acceptable but the two halves of the resulting dinucleotide binding domains are evolutionarly uncorrelated. The NH2-terminal mononucleotide binding domain is less variable than the COOH-terminal half, probably because it involves the binding of the ADP moiety of NAD(P) invariant in all examined systems. There is evidence to postulate that evolutionary pathways for NAD(P)-dependent enzymes are both divergent and convergent. In fact, nearly all combinations of similarity/dissimilarity in overall fold, cofactor conformation, and cofactor binding pocket structural characteristics for each enzyme pair examined are possible. The NAD(P)-dependent enzymes apparently provide a canonical example of an evolutionary principle that “anything goes.” © 1997 Wiley-Liss Inc. 相似文献
13.
Ji-Hong Bai Hao-Jing Wang De-Shan Liu Hai-Meng Zhou 《Journal of Protein Chemistry》1997,16(8):801-807
The kinetics of thermal inactivation of rabbit muscle lactate dehydrogenase at different temperatures has been studied using the kinetic method for the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [Adv. Enzymol. Relat. Areas Mol. Biol. (1988), 61, 381–436]. The results show that thermal inactivation of the enzyme is an irreversible reaction. Microscopic rate constants were determined for thermal inactivation of the free enzyme and the enzyme–substrate complex. The inactivation rate constant of the free enzyme is much larger than the rate constant of the enzyme–substrate complex. The results suggest that the presence of the substrate has a certain protective effect against thermal inactivation of the enzyme. 相似文献
14.
McCorvie TJ Liu Y Frazer A Gleason TJ Fridovich-Keil JL Timson DJ 《Biochimica et biophysica acta》2012,1822(10):1516-1526
Deficiency of UDP-galactose 4'-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and no ability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD(+). p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two α-helices that contain residues that interact with NAD(+). These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation. 相似文献
15.
Jingjing GuoLulu Ning Hui RenHuanxiang Liu Xiaojun Yao 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012,1820(2):116-123
Background
Prion diseases are associated with a conformational switch for PrP from PrPC to PrPSc. Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD.Scope of review
MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native β-sheet and the conversion of S2-H2 loop to a 310 helix.Major conclusions
Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrPC to PrPSc.General significanceOur present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrPSc assemblies. 相似文献16.
Guodong Zhang Jian An Chen Takuji Tanaka 《Biochimica et Biophysica Acta - Proteins and Proteomics》2009,1794(6):968-975
The allosteric behaviour of Lactococcus lactis prolidase (Xaa-Pro dipeptidase) of this proline-specific peptidase was investigated where it was hypothesized that intersubunit interactions between a loop structure and three residues near the active site contributed to this behaviour. Seven mutant prolidases were constructed, and it was observed that the loopless mutant and His303 substitution inactivated the enzyme. Ser307 substitution revealed that this residue influenced the substrate binding, as judged from its kinetic constants and substrate specificity; however, this residue did not contribute to allostery of prolidase. R293S mutation resulted in the disappearance of the allosteric behaviour yielding a Hill constant of 0.98 while the wild type had a constant of 1.58. In addition, the R293S mutation suppressed the substrate inhibition that was observed in other mutants and wild type. The Km value of R293S was 2.9-fold larger and Vmax was approximately 50% less as compared to the wild type. The results indicated that Arg293 increased the affinity for substrates while introducing allosteric behaviour and substrate inhibition. Computer modelling suggested that negative charges on the loop structure interacted with Arg293 and Ser307 to maintain these characteristics. It was, therefore, concluded that Arg293, His303, Ser307 and the loop contributed to the enzyme's allosteric characteristics. 相似文献
17.
Bin Wen Lei Xu Yawei Tang Zhen Jiang Mengting Ge Li Liu Guoping Zhu 《The Journal of biological chemistry》2022,298(8)
The UV-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 photoproducts), can be directly photorepaired by CPD photolyases and 6-4 photolyases, respectively. The fully reduced flavin (hydroquinone, HQ) cofactor is required for the catalysis of both types of these photolyases. On the other hand, flavin cofactor in the semireduced state, semiquinone, can be utilized by photolyase homologs, the cryptochromes. However, the evolutionary process of the transition of the functional states of flavin cofactors in photolyases and cryptochromes remains mysterious. In this work, we investigated three representative photolyases (Escherichia coli CPD photolyase, Microcystis aeruginosa DASH, and Phaeodactylum tricornutum 6-4 photolyase). We show that the residue at a single site adjacent to the flavin cofactor (corresponding to Ala377 in E. coli CPD photolyase, hereafter referred to as site 377) can fine-tune the stability of the HQ cofactor. We found that, in the presence of a polar residue (such as Ser or Asn) at site 377, HQ was stabilized against oxidation. Furthermore, this polar residue enhanced the photorepair activity of these photolyases both in vitro and in vivo. In contrast, substitution of hydrophobic residues, such as Ile, at site 377 in these photolyases adversely affected the stability of HQ. We speculate that these differential residue preferences at site 377 in photolyase proteins might reflect an important evolutionary event that altered the stability of HQ on the timeline from expression of photolyases to that of cryptochromes. 相似文献
18.
Clifford S. Morrison William B. Armiger David R. Dodds Jonathan S. Dordick Mattheos A.G. Koffas 《Biotechnology advances》2018,36(1):120-131
Industrial enzymatic reactions requiring 1,4-NAD(P)H2 to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H2 from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H2-dependent enzyme reactions of interest to the industrial biocatalysis community. 相似文献
19.
Bao-Jian Zhu Qiu-Ning Liu Li-Shang Dai Lei WangYu Sun Kun-Zhang LinGuo-Qing Wei Chao-Liang Liu 《Gene》2013
The complete mitochondrial genome (mitogenome) of Diaphania pyloalis (Lepidoptera: Pyralididae) was determined to be 15,298 bp and has the typical gene organization of mitogenomes from lepidopteran insects. It consists of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A + T-rich region. The A + T content of this mitogenome is 80.83% and the AT skew is slightly positive. All PCGs are initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which is initiated by CGA. Only the cox2 gene has an incomplete stop codon consisting of just a T. All the tRNA genes display a typical clover-leaf structure of mitochondrial tRNA. The A + T-rich region of the mitogenome is 332 bp in length, including several common features found in lepidopteran mitogenomes. Phylogenetic analysis showed that the D. pyloalis is close to Pyralididae. 相似文献
20.
Shirley Y. Lee Lester Pullen Daniel J. Virgil Carlos A. Castañeda Dulith Abeykoon Daniel N.A. Bolon David Fushman 《Journal of molecular biology》2014
Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. Both the two null mutants (I30A and L43A) were less stable to temperature-induced unfolding in vitro than wild type (WT) but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to WT. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high-molecular-weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high-molecular-weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings, we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation. 相似文献