Probing the Active Site of a Diels−Alderase Ribozyme by Photoaffinity Cross-Linking

2008 ◽  
Vol 130 (27) ◽  
pp. 8594-8595 ◽  
Author(s):  
Richard Wombacher ◽  
Andres Jäschke
Keyword(s):  
Active Site ◽  
Cross Linking

scholarly journals Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride

1991 ◽  
Vol 277 (1) ◽  
pp. 207-211 ◽  
Author(s):  
Y Z Ma ◽  
C L Tsou
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Cross Linking ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Original Activity ◽  
Limited Region ◽  
Low Concentrations ◽  
Two Stages ◽  
Inhibited Enzyme

The inactivation and unfolding of lactate dehydrogenase (LDH) during denaturation by guanidinium chloride (GuHCl) under diverse conditions have been compared. Unfolding of the native conformation, as monitored by fluorescence and c.d. measurements, occurs in two stages with increasing GuHCl concentrations, and the inactivation approximately coincides with, but slightly precedes, the first stage of unfolding. The enzyme is inhibited to about 60-70% of its original activity by cross-linking with glutaraldehyde or in the presence of 1 M-(NH4)2SO4, with its conformation stabilized as shown by the requirement for higher GuHCl concentrations to bring about both inactivation and unfolding. Low concentrations of GuHCl (0.2-0.4 M) activate the cross-linked and the (NH4)2SO4-inhibited enzyme back to the level of the native enzyme. For the enzyme stabilized by (NH4)2SO4 or by cross-linking with glutaraldehyde, inactivation occurs at a markedly lower GuHCl concentration than that required to bring about its first stage of unfolding. It is concluded that the active site of LDH is situated in a limited region relatively fragile in conformation as compared with the molecule as a whole. The GuHCl activation of LDH stabilized in (NH4)2SO4 or by cross-linking with glutaraldehyde suggests that this fragility and consequently flexibility of the active site is required for its catalytic activity.

An intersubunit interaction at the active site of D-ribulose-1,5-bisphosphate carboxylase/oxygenase as revealed by cross-linking and site-directed mutagenesis

Biochemistry ◽  
1987 ◽  
Vol 26 (15) ◽  
pp. 4599-4604 ◽  
Author(s):  
Eva H. Lee ◽  
Thomas S. Soper ◽  
Richard J. Mural ◽  
Claude D. Stringer ◽  
Fred C. Hartman
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Bisphosphate Carboxylase

scholarly journals Site-Specific, Intramolecular Cross-Linking of Pin1 Active Site Residues by the Lipid Electrophile 4-Oxo-2-nonenal

2015 ◽  
Vol 28 (4) ◽  
pp. 817-827 ◽  
Author(s):  
Christopher D. Aluise ◽  
Jeannie M. Camarillo ◽  
Yuki Shimozu ◽  
James J. Galligan ◽  
Kristie L. Rose ◽  
...  
Keyword(s):  
Active Site ◽  
Cross Linking ◽  
Active Site Residues ◽  
Site Specific

Photochemical mapping of the active site of myosin

1992 ◽  
Vol 336 (1276) ◽  
pp. 55-61 ◽  
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Cross Linking ◽  
Detailed Structure ◽  
Atp Binding Site ◽  
Adenine Ring ◽  
Skeletal Myosin ◽  
Key Residues ◽  
Specific Labelling

The active sites of myosin from skeletal, smooth and scallop muscle have been partly characterized by use of a series of photoreactive analogues of ATP. Specific labelling was attained by trapping these analogues in their diphosphate forms at the active sites by either cross-linking two reactive thiols (skeletal myosin) or by formation of stable vanadate-metal ion transition state-like complexes (smooth muscle and scallop myosin). By use of this approach combined with appropriate chemistry, several key residues in all three myosins have been identified which bind at or near the adenine ring, the ribose ring and to the γ-phosphate of ATP. This information should aid in the solution of the crystal structure of the heads of myosin and in defining a detailed structure of the ATP binding site.

scholarly journals Dibromopropanone Cross-linking of the Phosphopantetheine and Active-site Cysteine Thiols of the Animal Fatty Acid Synthase Can Occur Both Inter- and Intrasubunit

1999 ◽  
Vol 274 (17) ◽  
pp. 11557-11563 ◽  
Author(s):  
Andrzej Witkowski ◽  
Anil K. Joshi ◽  
Vangipuram S. Rangan ◽  
Arnold M. Falick ◽  
H. Ewa Witkowska ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Active Site ◽  
Fatty Acid Synthase ◽  
Cross Linking ◽  
Active Site Cysteine ◽  
Cysteine Thiols

Heme Oxygenase Active-Site Residues Identified by Heme−Protein Cross-Linking during Reduction of CBrCl3†

Biochemistry ◽  
1998 ◽  
Vol 37 (9) ◽  
pp. 2889-2896 ◽  
Author(s):  
Angela Wilks ◽  
Katalin F. Medzihradszky ◽  
Paul R. Ortiz de Montellano
Keyword(s):  
Heme Oxygenase ◽  
Active Site ◽  
Heme Protein ◽  
Cross Linking ◽  
Active Site Residues ◽  
Protein Cross Linking

scholarly journals Nonnucleoside Inhibitor Binding Affects the Interactions of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with DNA

2004 ◽  
Vol 78 (7) ◽  
pp. 3387-3397 ◽  
Author(s):  
Elena N. Peletskaya ◽  
Alex A. Kogon ◽  
Steven Tuske ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Active Site ◽  
Cross Linking ◽  
Immunodeficiency Virus ◽  
Dntp Binding ◽  
Hiv 1

ABSTRACT Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the “open” configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

scholarly journals GSAP modulates γ-secretase specificity by inducing conformational change in PS1

2018 ◽  
Author(s):  
Eitan Wong ◽  
George P. Liao ◽  
Jerry Chang ◽  
Peng Xu ◽  
Yue-Ming Li ◽  
...  
Keyword(s):  
Conformational Change ◽  
Active Site ◽  
Cultured Cells ◽  
Cross Linking ◽  
Secretase Activity ◽  
Aβ Production ◽  
Insight Into ◽  
Double Cross

AbstractThe mechanism by which GSAP (γ-secretase activating protein) regulates γ-secretase activity has not yet been elucidated. Here, we show that knockout of GSAP in cultured cells directly reduces γ-secretase activity for Aβ production, but not for Notch1 cleavage, suggesting that GSAP may induce a conformational change contributing to the specificity of γ-secretase. Furthermore, using an active site directed photoprobe with double cross-linking moieties, we demonstrate that GSAP modifies the orientation and/or distance of PS1-NTF and PS1-CTF, a region containing the active site of γ-secretase. This work offers insight into how GSAP regulates γ-secretase specificity.

scholarly journals Structures of Human Transglutaminase 2: Finding Clues for Interference in Cross-linking Mediated Activity

2020 ◽  
Vol 21 (6) ◽  
pp. 2225
Author(s):  
Gi Eob Kim ◽  
Hyun Ho Park
Keyword(s):  
Neurodegenerative Disorders ◽  
Active Site ◽  
Drug Target ◽  
Structural Information ◽  
Transglutaminase 2 ◽  
Cross Linking ◽  
Targeted Drugs ◽  
Clinical Use ◽  
Structural Studies ◽  
Cellular Processes

Human transglutaminase 2 (TGase2) has various functions, including roles in various cellular processes such as apoptosis, development, differentiation, wound healing, and angiogenesis, and is linked to many diseases such as cancer. Although TGase2 has been considered an optimized drug target for the treatment of cancer, fibrosis, and neurodegenerative disorders, it has been difficult to generate TGase2-targeted drugs for clinical use because of the relatively flat and broad active site on TGase2. To design more specific and powerful inhibitors, detailed structural information about TGase2 complexed with various effector and inhibitor molecules is required. In this review, we summarized the current structural studies on TGase2, which will aid in designing drugs that can overcome the aforementioned limitations.

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