Angiotensin converting enzyme: substrate inhibition

Phosphate, borate, and Tris inhibit angiotensin converting enzyme (ACE), but HEPES buffer is inert. Measurements of substrate inhibition were made in HEPES buffer at pH 7.0 and 25 degrees C and 37 degrees C. Substrate inhibition was marked and goes to completion. A new equation for substrate inhibitions enables one, under favorable circumstances, to determine whether there is cooperativity in the binding of substrate to the inhibitory and active sites. Cooperativity does occur with ACE using Hipp-His-Leu as substrate. The kinetic parameters were measured (Km = 0.21 mM, K* = 0.65 mM at 37 degrees C). The enzyme concentration (1.94 X 10(-8) M) was determined by titration with lisinopril so that kcat (5 X 10(3) at 37 degrees C) could be determined. Using this value and the molecular weight the specific activity of ACE was calculated for different common buffers. The specific activity in HEPES calculated from Vmax was 33.7 units/mg at 37 degrees C.     

Structure-based substrate screening for an enzyme

Background  

Nowadays, more and more novel enzymes can be easily found in the whole enzyme pool with the rapid development of genetic operation. However, experimental work for substrate screening of a new enzyme is laborious, time consuming and costly. On the other hand, many computational methods have been widely used in lead screening of drug design. Seeing that the ligand-target protein system in drug design and the substrate-enzyme system in enzyme applications share the similar molecular recognition mechanism, we aim to fulfill the goal of substrate screening by in silico means in the present study.     

A new chromogenic substrate for angiotensin-converting enzyme

The compound para-nitrobenzyloxycarbonylglycyl-(S-4-nitrobenzo-2-oxa-1,3-diazole)-l-cysteinylglycine [NO₂ZGly(S-NBD)CysGly] with an absorption maximum at 423 nm is readily hydrolyzed by angiotensin-converting enzyme (EC 3.4.15.1, peptidlyldipeptide hydrolase) to yield the S-benzfurazan derivative of cysteinylglycine. An internal S → N shift occurs immediately to yield the N-benzfurazan derivative which in turn reacts with the sulfhydryl reagent 4,4′-dithiodipyridine to produce the mixed disulfide with an intense absorption at 461 nm. The maximum difference in molar absorptivity of 13,000 m⁻¹ cm⁻¹ occurs at 470 nm.     

Catechol-O-methyltransferase biochemical genetics: Human lymphocyte enzyme

Human erythrocyte (RBC) catechol-O-methyltransferase (COMT) is under genetic control. Experiments were performed to determine whether COMT in the human lymphocyte is regulated in parallel with RBC COMT. Supernatants of lymphocyte homogenates contained COMT activity. However, they also contained a potent COMT inhibitor, the effect of which could be negated by dilution. Lymphocyte COMT activity was maximal at a reaction pH of 7.7 and at a MgCl₂ concentration of 0.67mm. The apparent K _m value for 3,4-dihydroxybenzoic acid, the catechol substrate for the reaction, was 1.2×10^?5 m and that for S-adenosyl-l-methionine, the methyl donor, was 2.3×10^?6 m. An average of 48.3±3.3% (mean ± SEM) of the enzyme activity in crude lymphocyte homogenates from 3 subjects was removed by centrifugation at 100,000 g for 1 hr and was presumed to be membrane associated. The average COMT activity in lymphocytes isolated from blood of 23 randomly selected adult subjects was 14.0±1.2 units/10⁶ cells (mean ± SEM) or 913±69 units/mg protein. There was a significant correlation of relative RBC with relative lymphocyte COMT activity in these 23 subjects. The correlation coefficient was 0.733 (P<0.001) when lymphocyte enzyme activity was expressed per milligram of protein and 0.649 (P<0.001) when lymphocyte activity was expressed per 10⁶ cells. These results are compatible with the conclusion that the genetic polymorphism which regulates RBC COMT activity may also regulate the level of human lymphocyte COMT activity.     

Angiotensin-converting enzyme inhibitors: biochemical properties and biological actions

The review will cover the chemistry and biochemistry of angiotensin-converting enzyme inhibitors with emphasis on data published since the publication of previous reviews. The relative merits of each contribution will be evaluated, as well as their potential for leading to new discoveries. The biology of angiotensin-converting enzyme inhibitors will be brought up-to-date to give the reader an appreciation of the medical implications of this new type of antihypertensive agent.     

生物化学实验教学的现状分析与改革思路

现行的生物化学实验教学在“学习知识、验证理论”等方面是可取的,但这种以验证生化过程为主的实验,教学目的过于单一、教学方式高度“程式化”,已不能适应高校培养创新型人才的需要。应从培养学生的创新能力和探索精神出发,深化对生物化学实验教学的改革：一、重新认识实验教学的重要性;二、增加综合性、探索型实验,重点培养学生的创新能力;三、灵活运用多样化的教学形式和教学方法;四、建立科学的实验考核体系。     

Inorganic pyrophosphatase--a new enzyme label for biochemical analysis

Inorganic pyrophosphatase isolated from Escherichia coli has been proposed as a label in heterogeneous enzyme immunoassays. The enzyme is remarkably stable and insensitive to sodium azide. Enzyme-antibody conjugates were prepared with glutaraldehyde and purified by gel filtration. Enzyme activity was measured by means of a sensitive colour reaction between phosphomolybdate and malachite green. A 5-10-fold increase is sensitivity in terms of absorbance readings was observed compared to peroxidase-based assays. The colour change (yellow/greenish blue) inherent in the use of pyrophosphatase as the labelling agent is highly suitable for visual analysis.     

Recent trends in cell substrate considerations for continuous cell lines

Product development activity in the past five to ten years has reconstituted a version of an old debate on the safety assessment of biological products, namely whether the use of some types of continuous cell lines (CCLs) is appropriate in the preparation of some types of biological products. Since 1987, dozens of purified recombinant DNA products derived from CCLs have been developed and have received regulatory approval. In addition, several live attenuated and inactivated viral vaccines manufactured in CCLs were approved after thorough review of product safety and manufacturing issues. The current discussion revolves around the potential use of CCLs (human or not) to prepare purified protein subunit vaccines, such as for HIV, and the use of human CCLs to prepare purified protein products.     

Inhibition of angiotensin converting enzyme: mechanism and substrate dependence

The interaction of angiotensin converting enzyme with six metal-coordinating [(D-3-mercapto-2-methylpropanoyl)-L-Pro (captopril), N-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Pro (MK-422), N-(phenylphosphoryl)-L-Phe-L-Phe, N alpha-(3-mercaptopropanoyl)-L-Arg, N alpha-[1(S)-carboxy-3-phenylpropyl]-Ala-L-Lys, and N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly] and three dipeptide inhibitors (Gly-L-Trp, L-Phe-L-Arg, and L-Ala-L-Pro) was examined at pH 7.5 in the presence of 300 mM NaCl. Inhibition modes, apparent Ki [Ki(app)] values, and shapes of 1/v vs. [I] plots were found to vary with the substrate employed. All inhibitors except Phe-Arg were competitive with the substrate furanacryloyl (Fa)-Phe-Gly-Gly, while five of seven tested with Fa-Phe-Phe-Arg as substrate produced mixed patterns. Ki-(app) values for N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly, N-(phenylphosphoryl)-L-Phe-L-Phe, Gly-Trp, and MK-422 were 8.3-, 5.5-, 4.7-, and 2.6-fold lower, respectively, when Fa-Phe-Gly-Gly was substrate, compared with values measured with Fa-Phe-Phe-Arg. In contrast, Ki(app) values for Phe-Arg and (3-mercaptopropanoyl)-Arg were lower (2.8- and 2.2-fold, respectively) when Fa-Phe-Phe-Arg was the substrate. Plots of 1/v vs. [I] for most of the inhibitors were nonlinear, to an extent which was also substrate dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

ADAM33 enzyme properties and substrate specificity

ADAM33 is an asthma susceptibility gene recently identified through a genetic study of asthmatic families [van Eerdewegh, et al. (2002) Nature 418, 426-430]. To understand the function of the gene product, the recombinant metalloproteinase domain of human ADAM33 was purified and tested for its substrate cleavage specificity using peptides derived from beta-amyloid precursor protein (APP). A single Ala substitution at the P2 position of a 10-residue APP peptide, YEVHHQKLVF, yielded a 20-fold more efficient substrate. Terminal truncation studies identified a minimal nine-residue core (P5-P4') important for ADAM33 recognition and cleavage. Full positional scanning of the 10-mer peptide using the 19 naturally occurring l-amino acids (excluding Cys) revealed a substrate specificity profile. A strong preference for Val or Ile at P3, Ala at P2, and Gln at P1' was observed. The substrate binding model based on the X-ray structure of the ADAM33-inhibitor complex supported the observed substrate specificity profile. On the basis of this, an improved substrate was designed and a fluorescence resonance energy transfer (FRET) assay was developed using a fluorogenic derivative of this substrate. Kinetic studies confirmed that the best substrate, FRET-P2 [K(Dabcyl)YRVAFQKLAE(Edans)K], was approximately 100-fold more efficient than the wild-type APP peptide substrate, with a k(cat)/K(m) value of (3.6 +/- 0.1) x 10(4) s(-)(1) M(-)(1). Using this substrate and the FRET assay, ADAM33 enzyme activity and thermal stability were characterized. ADAM33 dependence on buffer conditions, detergents, and temperature was examined, and optimal conditions were defined. Accurate K(i) values for tissue inhibitors of metalloproteinase and small molecule compounds were obtained.     

Activity dependent characteristics of fast and slow muscle: biochemical and histochemical considerations

The effects of denervation and hindlimb suspension induced disuse on concentrations of ATP, phosphocreatine (PC), and fiber type profile were investigated in slow twitch soleus and fast twitch extensor digitorum longus (EDL) muscles. The results show that the soleus and EDL muscles differ in their dependency on loadbearing as a stimulus for maintaining normal energy metabolism and the biochemical and morphological characteristics of muscle fibers. As determined by R-P methodology, suspension reduced ATP and PC concentrations of the soleus to 26% and 56%, respectively, while, in EDL only, PC is reduced to 71% of control with no change in ATP. Both muscles, however, show identical losses in ATP and PC following denervation. The energy charge, an indicator of Pi availability in muscle was reduced significantly in both denervated muscles to 82% and 85% in soleus and EDL, respectively. No significant reduction of the energy charge was seen in the muscles from suspended rats. Thus, in parallel with the indirect regulation through muscle loadbearing, the nerve can effectively modulate the levels of high-energy phosphates more directly by some regulatory mechanisms independent of muscle type. Denervation and suspension disuse increased the proportion of type 2 fibers in the soleus with a concomitant decrease in type 1 fibers and a relative rise in the number of very small diameter fibers. The EDL showed only variation in fiber size.     

Ubiquitin is a novel substrate for human insulin-degrading enzyme

Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β, peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (k_cat = 2 s^− 1) followed by a slow cleavage between residues 72 and 73 (k_cat = 0.07 s^−  1), thereby producing the inactive 1-74 fragment of Ub (Ub1-74) and 1-72 fragment of Ub (Ub1-72). IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼ 90-fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG <  0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE.